UC-NRLF 




A LABORATORY GUIDE IN 
BACTERIOLOGY 



THE UNIVERSITY OP CHICAGO PRESS 
CHICAGO, ILLINOIS 



THE BAKER AND TAYLOR COMPANY 

HEW TORE 



THE CAMBRIDGE UNIVERSITY PRESS 

LONDON 

THE MARUZEN-KABUSHIKI-KAISHA 

TOKYO, OSAKA, KYOTO, FUKUOIA, SSHDAI 

THE MISSION BOOK COMPANY 



A LABORATORY GUIDE 

IN 

BACTERIOLOGY 



FOR THE USE OP STUDENTS, TEACHERS 
AND PRACTITIONERS 



BY 



PAUL G. HEINEMA 3P4p.;';$ ' : \\ \ 



THIRD REVISED EDITION 




THE UNIVERSITY OF CHICAGO PRESS 
CHICAGO, ILLINOIS 



- ' . COPYRIGHT 1905 BY 
'> \ THE UNIVERSITY OF CHICAGO 

All Rights Reserved 

First Edition June 1905 

Second Edition June 1911 

Second Impression October 1911 

Third Impression October 1913 

Third Edition September 1915 

Second Impression September 1916 

Third Impression July 1919 
Fourth Impression February igaa 



Composed and Printed By 

The University of Chicago Pre 

Chicaso. Illinois. U.S.A. 



PREFACE TO FIRST EDITION 

The considerations which led the writer to add this 
laboratory guide of bacteriology to the number of such 
guides already in- existence were of various nature and 
may be briefly set forth here. 

Probably no branch of biological science has ad- 
vanced so rapidly during the past few years as the 
science of bacteriology, and it is difficult even for an 
active laboratory worker to keep abreast of this ad- 
vance. A textbook or guide fixes the status of the 
science at the time of its writing, but almost before it 
leaves the press it becomes antiquated. Revisions, 
corrections, and additions are necessary at short 
intervals in order to keep a publication of this kind 
approximately up to date. There is, therefore, almost 
at any given moment room for a new publication to fill 
the want of a progressive instructor for a guide that 
gives the latest accepted rules and practices of the 
laboratory. The value of such a publication will 
be enhanced by a plan and arrangement of sufficient 
flexibility and latitude to allow the instructor and the 
student to enter such additions and corrections as serve 
to bridge over the time between editions. 

Medical students entering on a course in bacteri- 
ology often have had too little previous laboratory 
training in methods of precision. It is a matter of 
importance for the instructor to put himself in the atti- 
tude of mind of the student and to try to appreciate 
his difficulties in understanding details. Many of 



47638 



vi PREFACE TO FIRST EDITION 

the pieces of apparatus employed in a bacteriological 
laboratory are novel even to the student trained in 
chemistry and biology, and it has been thought best 
to exhibit these, to the smallest detail, by means of 
illustrations a feature not sufficiently considered in 
other guides. 

The formulae for stains and the methods of staining 
have not been collected in one chapter, as is usually the 
practice, because this tends to confuse the student. 
They are described during the progress of the course, as 
occasion offers to put them to practical use. 

Culture-description charts have not been included in 
this volume. A beginner naturally makes incomplete 
descriptions and many alterations, and thus defaces the 
book and impairs its future utility. A sufficient num- 
ber of loose charts perforated for binding should be 
furnished to the student at a nominal figure. 

A point of inestimable importance is how best to 
stimulate the student to consult textbooks, special 
monographs, and .other references, as often and as 
freely as possible. This guide has been written with 
the aim of not only not interfering in any manner with 
the reading, through including such points and char- 
acteristics as might make a textbook superfluous in the 
judgment of the inexperienced, but also of making it 
necessary for the student to read the best textbooks 
with freedom and understanding. Cultural and mor- 
phological features are left entirely to the actual ob- 
servation of the student, supplemented by instruction 
and the reading of textbooks. 

The course, as outlined, is identical with the medical 
course given at the University of Chicago, with a few 



PREFACE TO FIRST EDITION vii 

additional chapters which may be used during courses 
for non-medical students. A chapter containing a 
fairly complete list of formulae for culture media 
employed in advanced work has been added with the 
object of making them easily accessible to those engaged 
in advanced work. 

The laboratory guides of Novy, Eyre, Frost, 
Gorham, Kanthack and Drysdale, and Connell, and 
the American edition of the Manual of Bacteriology 
of Muir and Ritchie have been freely consulted. I 
take this occasion of expressing my deep gratitude to 
Professor Edwin O. Jordan and Dr. Norman MacLeod 
Harris for their invaluable help and suggestions in the 
preparation of this guide. 

PAUL G. HEINEMANN 
CHICAGO, ILL. 
June, 1905 



PREFACE TO SECOND EDITION 

The influence of applied bacteriology is extending 
rapidly, and while formerly the science of micro- 
organisms was chiefly confined to the medical field, it 
has become of vast industrial importance. The gigan- 
tic interests of fermentation industries, of the dairy, of 
agriculture, of municipal sanitation, and of water puri- 
fication are largely controlled by bacteriologists. A 
knowledge of the principles of bacteriology is becoming 
more and more desirable to the student of general 
science, and teachers of domestic science in some 
institutions are required to take an elementary course. 

The second edition of this laboratory guide has been 
revised and enlarged with the view of meeting modern 
requirements. The first edition was devoted chiefly to 
medical bacteriology. This has been partly rewritten, 
and in addition courses in general bacteriology, in the 
bacteriology of water, in the bacteriology of milk, in 
soil bacteriology, and a course on molds, yeasts, and 
acetic-acid bacteria have been outlined. This change of 
plan necessitated an entirely new arrangement of the 
material. The preparation of culture media, of stains, 
etc., has been incorporated in a separate part, and 
reference to this part is made at the beginning of each 
special part. The course in the bacteriology of water 
is calculated to be combined with the physical, chemical, 
and microscopic examination, the student being thus 
prepared for sanitary water analysis. Similarly, the 
course on the bacteriology of milk is to be given in 

ix 



X PREFACE TO SECOND EDITION 

connection with the physical and chemical examina- 
tion of milk. 

The author hopes that, by the radical changes indi- 
cated, the usefulness of the book may be extended, and 
that the study of applied bacteriology may be en- 
couraged. I am under special obligations to Professor 
Edwin O. Jordan for advice and suggestions as to the 
general plan of the book, to Dr. Mary Hefferan for 
critical reading of the manuscript and the proof, and 
to Dr. R. E. Buchanan for valuable advice in outlining 
the course in soil bacteriology. 

PAUL G. HEINEMANN 
June, 1911 



PREFACE TO THIRD EDITION 

In this third edition the general plan of the book 
has not been changed. The progress of science 
necessitated alterations and additions which have 
been incorporated hi the text. A fermentation chart 
of cardboard has been added to the colony counter 
in the back cover. 

The author is under obligations to Professors 
Edwin O. Jordan and Norman MacLeod Harris for 
valuable suggestions. 

PAUL G. HEINEMANN 

June, 1915 



INTRODUCTION 

The advent of bacteriology into the realm of the 
biological sciences not only brought with it a new con- 
ception of the nature of many complicated phenomena, 
such as fermentation and disease, but also placed in the 
hands of experimental workers a new tool. The 
method of sterilization, of asepsis, made it possible for 
the first time to attack problems hitherto incapable of 
solution, or even of approach. This development 
of bacteriological technic, of rigid and undeviating 
adherence to definite rules and principles, is not likely 
to be passed over lightly by the historian of nineteenth- 
century science. The art of practical medicine and 
theoretical medical research alike owe much of their 
recent brilliant success to a ready adoption of the new 
method. 

At the present time an active campaign is being 
set on foot by public health authorities against several 
widespread and serious diseases of the human race. 
In various parts of the world malaria, tuberculosis, and 
typhoid fever are being fought energetically and with 
much success. In these systematic and organized 
movements the resources of bacteriology are being 
utilized as never before, and a full understanding of 
technical procedure and devices is deemed essential by 
all workers in this subject. The problems of water- 
supply and sewage disposal, of urban infantile mortal- 
ity, and of the control of contagious diseases are all 
bound up with the intelligent application of bacterio- 
logical methods. 



xii INTRODUCTION 

In the almost untilled field of industrial bacteri- 
ology there is need for a fuller appreciation of the 
value of bacteriological methods and principles. Many 
great industries are based wholly upon the proper 
selection and adaptation of micro-organisms, and a 
timely and discriminating utilization of their products. 
Loose and empirical methods have been in force in the 
past, but these must eventually give way to a more 
precise and truly bacteriological technic. 

Agricultural bacteriology is just now much in the 
public eye, and it would be gratuitous to prophesy 
the results that may reasonably be anticipated in this 
direction. Here again crude, rule-of-thumb, "prac- 
tical " ways of doing things are being supplanted by the 
scientific, the reasoning, and the precise. 

To the student, whether in medical, hygienic, or 
industrial bacteriology, proper technical methods of 
work must always have a peculiar value, since without 
their aid advance is impossible, and stumbling and 
disastrous missteps are certain. A comprehensive out- 
line of modern bacteriological methods, therefore, is a 
necessary adjunct to obtaining a true and full under- 
standing of the underlying principles and tendencies 
of the science. The technic of bacteriology is one 
of its greatest contributions to both science and art, 
and the use of so valuable and simple a tool should be 
mastered not only by the biological teacher and in- 
vestigator, but by practical workers in medicine, 
hygiene, and many other fields. 

EDWIN O. JORDAN 



TABLE OF CONTENTS 



PART I. BACTERIOLOGICAL TECHNIC 

Section i. Laboratory Rules 

Section 2. General Directions 

Preparation and Cleaning of Glassware 
Methods of Sterilization .... 

Preparation of Culture Media . . 

Preparation of Staining Solutions 

The Microscope ....... 45 

Scheme for Routine Study of Bacteria . 50 
Method of Describing Cultures ... 53 
Directions for Filling out Culture Charts 58 



Section 
Section 
Section 
Section 
Section 
Section 
Section 9. 
Section 10. 



PAGE 

3 

4 

8 

10 

18 

43 



PART II. GENERAL BACTERIOLOGY 

Section i. Preparation of Culture Media ... 69 
Section 2. Collecting and Cultivating Micro-organ- 
isms from the Air 70 

Section 3. Study of Molds, Yeasts, and Torulae . 78 
Section 4. Bacteriological Examination of Water, 

Air, and Milk . 84 

Section 5. Exercises on Infection and Sterilization . 91 
Section 6. Influence of Disinfectants, Light, and 

Heat on the Growth of Micro-organisms 94 

Section 7. Study of Chromogenic Bacteria ... 97 

Section 8. Study of Micrococci 100 

Section 9. Study of Intestinal Bacteria . . . . 101 

PART III. IMPORTANT PATHOGENIC BACTERIA 

Section i. Preparation of Culture Media . . . 105 

Section 2. The Pyogenic Group 105 

Section 3. The Group of Colon-Typhoid Bacilli . 112 

Section 4. The Proteus Group . . .. . . . 121 

Section 5. The Capsulated Group 123 

Section 6. The Diphtheria Group 124 



xiv 



TABLE OF CONTENTS 



PAGE 

Section 7, The Hemorrhagic Septicemia Group . . 127 

Section 8. The Anthrax Group . . . . , * 127 

Section 9. The Spirillum Group 129 

Section 10. The Group of Acid-proof Bacilli . . . 131 

Section n A and B. Miscellaneous Organisms . 132, 133 

Section 12. Pathogenic Trichomycetes . . . . 134 

Section 13. The Group of Anaerobic Bacilli . . . 134 
Section 14. Isolation of Unknown Bacteria from a 

Mixture 139 

PART IV. THE BACTERIOLOGICAL EXAMINATION OF WATER 
AND SEWAGE 
Section i. Preparation of Culture Media and of 

Dilution Flasks 144 

Section 2. Bacteriological Examination of Water . 145 

Section 3. Examination of Sewage 148 

Section 4. Determination of Anaerobes in Sewage . 149 
Section 5. A Study of B. coli and Streptococci . . 149 
Section 6. Isolation of B. typhosus from Water . 150 
Section 7. Study of Reaction of Bacteria on Neutral- 
red Broth ... . . . . . . 150 

PART V. THE BACTERIOLOGICAL EXAMINATION OF MILK 

Section i. Preparation of Culture Media and of 

Dilution Flasks . 154 

Section 2. Quantitative Bacteriological Examina- 
tion of Milk 154 

Section 3. Examination of Market Milk for Tubercle 

Bacilli iSS 

Section 4. A Study of the Acid Fermentation of Milk 155 

Section 5. Determination of B. coli and Streptococci 

in Milk 157 

Section 6. Study of the Effects of Pasteurization 

and So-called Sterilization of Milk . . 158 

Section 7. A Qualitative and Quantitative Study of 

Anaerobes in Milk 158 

Section 8. A Study of Some Organisms Causing Ab- 
normal Feonentations in Milk . . . 159 



TABLE OF CONTENTS 



xv 



Section 9 



Section 
Section 
PART VI. 
Section 



Section 
Section 
Section 
Section 
Section 
Section 
Section 
Section 



Examination of Milk for Molds and 
Yeasts 160 

10. Examination for Leukocytes in Milk . 160 

11. A Study of Groups of Bacteria in Milk . 162 
THE BACTERIOLOGICAL EXAMINATION OF SOIL 

1. Quantitative Determination of Bacteria 
and Spores in Soil 165 

2. A Study of the Peptonization of Proteins 

by Soil Bacteria 168 

3. The Formation of Amido Compounds and 
Ammonia 170 

4. The Formation of Nitrites from Ammonia 
and Isolation of Nitrite Bacteria . . . 171 

5. The Formation of Nitrates from Nitrites 

and Isolation of Nitrate Bacteria . . . 174 

6. The Assimilation of Free Atmospheric 
Nitrogen and Isolation of the Bacteria . 175 

7. The Reduction of Nitrates to Nitrites and 
Isolation of the Bacteria 177 

8. The Reduction of Nitrates to Free Nitro- 
gen .. 177 

9. Growing Legumes in Sand and in Sand 
Inoculated with Legume Bacteria 



. 178 

PART VII. MOLDS, YEASTS, TORULAE, AND ACETIC-ACID 
BACTERIA 

Section i. Preparation of Culture Media . . . 181 

Section 2. A Study of Molds 182 

Section 3. A Study of Yeasts 185 

Section 4. Examination of Baker's Yeast . . . 190 

Section 5. Examination of Yeast of Salt-rising Bread 191 

Section 6. A Study of Torulae 192 

Section 7. A Study of Acetic-Acid Bacteria . . . 192 

APPENDIX 

Dilution Tables 197 

Table of Weights and Measures 198 

Table of Centigrade and Fahrenheit Thermometers . 198 

INDEX 203 



PART I 
BACTERIOLOGICAL TECHNIC 



\ 



SECTION i 
LABORATORY RULES 

1. Familiarize yourself with the laboratory rules. 
Upon their careful observance depend good work and 
your own safety. 

2. Food must not be eaten in the laboratory; pencils, 
labels, or fingers must not be moistened with the 
tongue. 

3. If any portion of a culture is spilt by accident 
upon the desk or floor, it should be covered immediately 
with a germicide (HgCl 2 1:1000, or carbolic acid in 
5 per cent solution). After this germicide has acted 
for 10 or 15 minutes, wipe it up and throw the cloth or 
paper into a waste jar. 

4. In case the hands should come in contact with 
infectious material, they should be washed with one 
of the above mentioned germicides, and then scrubbed 
with soap and water. 

5. The platinum needles used in making cultures 
should be sterilized in a flame before and after use, and 
before they are laid down. When the needles are 
covered with viscous material, as milk, for instance, 
they should be held at the side of the flame until dry 
before being sterilized. This will avoid the danger of 
scattering infectious material about the desk. 

6. All possible care should be observed in handling 
apparatus, etc. Solid material should not be put into 
sinks. Burned matches, paper, cotton, broken glass, 

3 



;$,, LABORATORY GUIDE IN BACTERIOLOGY 

etc., should be put into crocks and not on the floor or 
into the sink. 

7. Discarded cultures should be killed in the auto- 
clave (5 minutes at 120 C.) before being emptied into 
the crocks. 

8. See that the air inlets of Bunsen burners are open 
before lighting, and relight if the flame strikes back. 

9. Always return stockbottles to the proper places 
on the shelves. 

10. At the close of the day's work the desks should 
be washed off with corrosive sublimate, and the hands 
cleaned by thorough washing. 

11. Before leaving the laboratory, see that the gas 
is shut off under all apparatus, that water faucets are 
closed, and that all glassware, etc., is replaced in the 
lockers. Culture tubes containing media or cultures 
should be replaced in their proper places, in order to 
avoid settling of dust or other foreign material on the 
stoppers. Dust and air draughts are frequently the 
cause of contaminations, and in order to avoid these 
the utmost cleanliness should be observed. A bacteri- 
ological laboratory should present an orderly appear- 
ance at all times. 

SECTION 2 
GENERAL DIRECTIONS 

The following directions should be followed in all 
the work outlined in this guide: 

i. After obtaining the key to a locker, the student 
should examine the outfit, check all apparatus, and see 
that everything is in good condition. 



BACTERIOLOGICAL TECHN^C- ; i ' V ' ; ; $ 

2. The student should familiarize himself with the 
program before him for each day, as this will facilitate 
intelligent and systematic work. 

3. The student should procure description charts 
and fill them out carefully according to directions. 

4. Store cover slips in a stender dish and cover 
them with alcohol. A soft linen cloth should be used 
for cleaning. 

The following outfit will be needed for each course. 
Additions to this outfit will be designated as required 
in the respective courses. 

200 culture tubes. 

20 potato tubes. 

12 fermentation tubes. 

20 petri dishes. 

3 Erlenmeyer flasks, one 1,000 c.c., two 500 c.c. each. 

2 glass funnels, one 4 inches, one 6 inches. 

4 bottles for staining fluids, 
i balsam bottle. 

i stender dish. 

3 staining dishes. 
i saltcellar. 

1 glass rod. 

2 platinum needles. Turn the end of one of the needles 

around a sharp pencil point so as to form a closed loop. 
8 tin cups or glass tumblers, the bottoms of which should be 
covered with a layer of cotton. 

4 wire baskets. 

i Bunsen burner. 

i saucepan and cover, or better, a double boiler. 

3 graduates, one 500 c.c., one 100 c.c., and one 10 c.c. 
i pinchcock. 

i pipette and hose attached, 
i magnifier (hand lens), 
i tripod. 



: ', 'I $.': ; r ','IABORATO'RV GUIDE IN BACTERIOLOGY 




Wire Basket 



Retort Stand 



Bottle for 
Staining Fluid Fermentation Tube 





Bunsen Burner 
FIG. * 



Culture Tube Pipette 



BACTERIOLOGICAL TECJINI^ ,' : ' 




Petri Dish 




Erlcnmeyer Flask 





Balsam Bottle 



Magnifier 
(Hand Lens; 




Tripod 




; Staining Dishes 




Saltcellar 




Slender Dish 



FIG. i 



: ' ; LABORATORY GUIDE IN BACTERIOLOGY 



i retort stand with three rings. 

i thermometer in case. 

i box matches. 

40 grams pepton. 

1 20 grams gelatin. 

50 grams agar. 

6 sheets filter paper. 

50 glass slides. 

1 camel's-hair brush. 
50 cover glasses. 

2 towels. 

i tube brush. 

50 labels. 

i box for slides. 

3 hollow-ground slides. 
i glass pencil. 

1 pair forceps. 

2 pairs cover-slip forceps. 

SECTION 3 
PREPARATION AND CLEANING OF GLASSWARE 

Culture tubes, flasks, fermentation tubes, and petr 
dishes must be free from organic matter, acids, anu 
alkalis. They should be cleaned as follows: 

1. Immerse them in a vessel containing soapsuds or 
soap powder, boil 10 to 15 minutes, then clean them 
with a tube brush. Or immerse them for an hour in a 
solution of potassium bichromate and sulphuric acid: 

Potassium bichromate ................ 60 parts 

Water .............................. 300 parts 

Concentrated sulphuric acid ........... 460 parts 

The sulphuric acid should be added slowly with con- 
stant stirring. 

2. Rinse in tap water. 



BACTERIOLOGICAL TECHNIC 



3. Again use tube brush, and soap and water if 
necessary. 

4. Rinse again in water to remove every trace of 
acid or soap. 

5. Place the tubes in a wire basket, mouth down, and 
heat in a hot-air sterilizer for 20 minutes or longer 
until dry. 

All other glassware should be treated in the same 
manner, excepting fermentation tubes, which should 
not be heated in the hot-air oven, as this would be 
likely to cause breakage. 

The tubes should be plugged with cotton. Non- 
absorbent cotton is suitable for this purpose. The 
cotton plug allows free communication with the air, 
admitting oxygen, which is necessary for the growth 
of many bacteria; at the same time the admitted air is 
filtered germ-free and contamination of cultures is 
avoided. 

Various methods for plugging tubes are employed 
in different laboratories. The simplest method is as 
follows: Take a small amount of cotton and push it 
gently into the tube with a glass 
rod. The cotton should reach 
into the tube for about J of an 
inch and be sufficiently firm to 
support the weight of the tube 
(Fig. 2). The cotton may also be 
rolled into a cylinder of thickness 
equal to that of the tube and then 
pushed into the mouth. 

The plugged culture tubes 
should be placed in a hot-air oven plugged Culture Tube 




10 LABORATORY GUIDE IN BACTERIOLOGY 

at a temperature of 150 C. for about 30 minutes, or 
until the plugs are slightly browned. The tubes are 
not necessarily sterile, but the plugs have become set 
so as to fit the mouth of the tube, and may be removed 
and replaced readily. 

SECTION 4 
METHODS OF STERILIZATION 

Sterilization is the process of removing all living 
organisms. This may be accomplished by heat, by 




FIG. 3 
Berkefeld Filter 

a. Berkefeld filter d. Intercepting flask 

b. Filtered liquid e. Connection with aspirator 

c. Side tube with cotton filter /. Rubber hose 



BACTERIOLOGICAL TECHNIC II 

certain chemicals, or by filtration. Chemicals are 
used chiefly for sterilizing the skin, surgical instru j 
ments, and cultures which have been accidentally 
spilt. Filters for sterilization are made of some porous 
material, either infusorial earth or unglazed porcelain. 
Substances which may be injured by heat are sterilized 
in this manner. Positive or 
negative pressure is necessary 
for this kind of sterilization 

(Fig. 3). 

Sterilization by dry heat. 

Sterilization by dry heat is 
applicable to the steriliza- 
tion of most glassware. This 
method of sterilization is car- 
ried out by means of hot-air 
sterilizers (Figs. 4 and 0. FlG -4 

r , . M. Koch's Hot-Air Sterilizer 

These hot-air sterilizers are 

boxes with double walls of sheet iron. The bottom 
shelf should always be covered with a piece of asbes- 
tos, to prevent heating the apparatus too rapidly. 
The temperature is maintained at 160 or more for one 
hour. The flame enters a hole provided at the bottom 
of the box. Care should be taken to avoid the possi- 
bility of the flame becoming luminous, otherwise the 
glassware will be covered with soot. 

Culture media and all substances liable to be in- 
jured by heat of 160 C. or over must be sterilized by 
the application of moist heat. Experience has taught 
that hot steam has greater germicidal powers than air 
of the same temperature. Hot steam, therefore, is the 
most common means of sterilizing culture media. 




12 LABORATORY GUIDE IN BACTERIOLOGY 

Steam is applied in two ways. The first method is 
that of exposing media to steam of 100 C. for 20 min- 
utes. This is done in an apparatus generally known as 
the Arnold steam sterilizer. The usual form is illus- 
trated in Figs. 6 and 7. Fig. 6 shows the appearance 




FIG. 5 
Lautenschlager Hot-Air Sterilizer 

of the ordinary form with the hood off. Fig. 7 shows 
the inside arrangement. The two compartments, a 
and b, are connected by small holes, and a certain 
amount of water has to be kept here. The water is 
brought to a boil and the steam rises through a number 
of holes in the bottom (c) into the chamber (d). The 
steam condenses at the top and returns between two 



BACTERIOLOGICAL TECHNIC 



sheet-copper walls (e, e) to the large compartment 
(b). Larger forms on the same principle are in use 
(Fig. 8). 

The media to be sterilized are placed in the large 
chamber (d, Fig. 7). The water is then heated until 
steam is generated, and the action of the steam on the 
media is continued for 20 minutes from the time steam 




FIG. 6 

Arnold Steam Sterilizer 
The hood is taken off and 
the door opened, showing 
inside arrangement 



FIG. 7 
Arnold Steam Sterilizer 

a. Inner water compartment 

b. Outer water compartment 

c. Perforated bottom 
d. Sterilizing chamber 

e. Sheet-copper walls 



begins to rise. This process is repeated on two succeed- 
ing days, so that the media have been exposed to the 
steam for three days. On the first day all vegetative 
forms are killed. The media are then kept at room or 
incubator temperature, so that spores which may be 
present and are not killed by the first exposure may 
develop into vegetative forms and be killed by the 
second exposure. If after this any spores should 



14 LABORATORY GUIDE IN BACTERIOLOGY 

survive, they will develop in the next 24 hours, and the 
third exposure to steam will complete sterilization. 

Sterilization is accomplished in a shorter time by 
the use of steam under pressure. The autoclave is the 
usual apparatus used for this purpose. Certain bac- 
teria, some of which are widely distributed in nature, 




FIG. 8 
Arnold Sterilizer 

have the faculty of forming spores. These spores are 
highly resistant to heat and do not lose their vitality 
either by boiling or by application of steam under 
ordinary atmospheric pressure. By adding the pres- 
sure of one atmosphere to ordinary pressure, the boiling 
point is raised to 121 .4 C. 120 C. is sufficient to kill 
all spores during an exposure of 5 minutes, if the media 
are in tubes. Larger amounts of media require a 
proportionately longer exposure. 

The autoclave consists of a strong cylinder made of 



BACTERIOLOGICAL TECHNIC 



iron. Some forms have a cover, others a door on one 
side. A basket, or a set of shelves, is on the inside. 
A gauge indicates the pressure and temperature. A 
safety valve opens automatically when the desired 
pressure is reached. Two 
forms are illustrated in 
Figs. 9 and 10. 

Before using the auto- 
clave the inside should be 
examined. It must be 
clean and contain a suffi- 
cient quantity of clean 
water. Water containing 
impurities is liable to foam 
up when boiling, wet the 
plugs, and ruin the media. 
If the lid is on top, it 
should be fastened care- 
fully by tightening the 
thumbscrews. In order 
to distribute the pressure 
of the lid uniformly the 
diametrically opposite 
screws should be tight- 
ened simultaneously. The 
valve should be open and 
left open until the steam 
has escaped for about one 
minute. Then the valve 
is closed and when the desired pressure has been 
reached, the gas should be turned down so as to 
maintain pressure for the requisite length of time. 




e 



FIG. 9 
Autoclave 

a. Steam valve d, d. Thumbscrews 

b. Safety valve . Bunsen burner 

c. Gauge and opening 



l6 LABORATORY GUIDE IN BACTERIOLOGY 



At the end of this period the gas is shut off and the 
pressure allowed to decrease gradually. The valve 
should not be opened, nor the lid removed, until 
atmospheric pressure has been restored, otherwise the 
sudden release of pressure would cause the media to 

boil suddenly and push 
the plugs out of place. 

When large autoclaves 
are used (Fig. 10) pro- 
vision must be made for 
proper circulation of air, 
or "air cushions" form 
and media will not be 
sterilized. If an aperture 
remains in the outlet the 
discharge of air is facili- 
tated. Attachment of a 
vacuum pump is often 
desirable to remove all air. 
Blood serum or egg 
media are the most diffi- 
cult to sterilize. The tem- 
perature of coagulation of 
these media is relatively 
low, and sudden heating 
causes the mass to break 
up, form bubbles, and be- 
come useless for cultural purposes. The Koch inspis- 
sator may be used, or, with certain precautions, the 
autoclave. The Koch inspissator (Fig. n) allows the 
tubes to rest in an inclined position and to be heated 
gradually to 75 C. This temperature is maintained 




BACTERIOLOGICAL TECHNIC 17 

for one hour. This process has to be repeated for five 
or six successive days, before sterilization is complete. 
If the autoclave is to be used, the tubes are placed 
in the autoclave in an inclined position. 
Good results are obtained by this method: 




FIG. n 
Koch Inspissator 

1. Close the lid and the steam outlet. 

2. Admit steam. After reaching 3 pounds pressure, 
keep this pressure for 5 minutes. 

3. Increase the pressure slowly to 5 pounds and 
keep there for 5 minutes. 



i8 LABORATORY GUIDE IN BACTERIOLOGY 

4. Increase to 10 pounds and hold for 5 minutes. 

5. Increase to 15 pounds and hold for 5 minutes. 

6. Open the steam outlet and keep at 15 pounds 
pressure for 20 minutes. 

It is advisable to sterilize the tubes again on the 
following day by slowly bringing the pressure to 15 
pounds with the steam outlet slightly open and keep- 
ing at this pressure for 20 minutes. 



SECTION 5 

PREPARATION OF CULTURE MEDIA 

EXERCISE I. PREPARATION OF DUNHAM'S PEPTON 
SOLUTION AND OF PEPTON BROTH (BOUILLON) 

1. Weigh the saucepan, measure into it 1,000 c.c. of 
tap water, and heat over a flame or a water bath. 

2. Dissolve in this, when hot, but not boiling, 10 
grams Witte's pepton. 

3. When dissolved, replace the evaporated amount 
of water and divide into two equal parts of 500 c.c. 
each. 

4. One-half is then filtered until perfectly clear, 
tubed, and sterilized in the autoclave for 5 minutes at 
i2oC. This is Dunham's Pepton Solution. 

5. Dissolve 1.5 grams extract of beef in the remain- 
ing 500 c.c 

6. Adjust the reaction with phenolphthalein papei 
or by titration against n. NaOH. 

NOTE. The reaction of culture media is a matter of vital 
importance. Bacteria, especially pathogenic bacteria, grow 
preferably in a medium which is neutral or slightly acid to 
phenolphthalein. The neutral point of litmus is about 2 per 



BACTERIOLOGICAL TECHNIC 19 

cent more alkaline than the neutral point of phenolphthalein, 
so that a medium which is neutral to phenolphthalem is about 
2 per cent alkaline to litmus. It has been found that about i 
per cent acid to phenolphthalein is the most favorable reaction 
for the growth of pathogenic bacteria. A medium of this reac- 
tion is still alkaline to litmus. 

7. After neutralization fill into an Erlenmeyer flask 
and autoclave for 10 minutes at 120 C. 

8. Keep the sterilized broth for 24 hours and then 
filter until clear and distribute into culture tubes, 
which have to be autoclaved again. 

NOTE. The reason for exposing broth to a heat of i2oC. 
twice is this: The solution contains substances which are pre- 
cipitated by heat and appear as a sediment after cooling. As it 
is important to have a perfectly clear broth in tubes, these sub- 
stances are precipitated by the first heating, and, if tubed later, 
the second sterilization will not affect the appearance of 8 the 
medium. 

For ordinary purposes it is sufficient to neutralize media by 
means of phenolphthalein paper. This is prepared by soaking 
filter paper in a i per cent solution of phenolphthalein in 50 per 
cent alcohol and then allowing the paper to become dry. A 2 per 
cent or 4 per cent solution of sodium hydrate is added to the 
medium to be neutralized until a faint, but decided, pink appears 
on phenolphthalein paper. 

A more precise method is as follows : Measure by means of a 
pipette 5 c.c. of the medium into a white porcelain evaporating 
dish, add 45 c.c. of distilled water and i c.c. of a i per cent solu- 
tion of phenolphathalein in 50 per cent alcohol. Heat the mix- 
ture to boiling and slowly add from a graduated burette i-2oth 
normal NaOH until a faint but decided and stable pink appears 
in the liquid. The amount of NaOH is read from the burette 
and the amount for neutralization of the whole volume calculated. 
It is desirable to make another titration after the NaOH has 
been added. The amount to be added to the medium has to be 
varied according to the reaction desired. If it is to be neutral, 



20 LABORATORY GUIDE IN BACTERIOLOGY 

the above proceeding will accomplish the object. If it is desired 
to have a medium which has a reaction of i per cent acid, a pro- 
portionate amount should be deducted from the total amount of 
NaOH calculated. 

Example. By reading the burette we find that it takes 3 c.c. 
i-2oth normal NaOH to neutralize 5 c.c. of the medium. This 
means that 60 c.c. i-20th n.NaOH will neutralize 100 c.c. medium, 
or 600 c.c. i-2oth n.NaOH will neutralize 1,000 c.c. medium. 
To find the requisite amount of n.NaOH divide the above figure 
by 20. Then 3 c.c. n.NaOH will neutralize 100 c.c. and 30 c.c. 
n.NaOH will neutralize 1,000 c.c. If the reaction is to be 
i per cent acid, deduct 10 c.c. from 30 = 20 c.c. If 20 c.c. 
normal NaOH are added to each liter the reaction should be i per 
cent acid. This should be ascertained by a second titration. 

A normal solution is the equivalent weight in grams (Gram- 
Molecule) dissolved in distilled water and made up to 1,000 c.c. 
In the case of monovalent chemicals the molecular weight is 
taken, if bivalent the molecular weight is divided by two, etc. 

^Vll media should be prepared with the utmost care 
and should be perfectly clear. 

EXERCISE 2. PREPARATION OF NUTRIENT AGAR-AGAR 

Agar-agar (or called simply "agar") is a watery 
extract of certain seaweeds found on the Pacific coast 
of Asia. A solution of agar containing about i . 5 per 
cent forms a firm jelly, which melts near the boiling 
point of water, and on cooling solidifies at about 39. 
Gelatin solidifies at much lower temperature, and can- 
not be kept solid at body temperature. The use of 
agar is, therefore, of great importance in the study of 
pathogenic bacteria, a large number of which prefer 
body temperature for growth. 

1. Weigh a saucepan, or, if available, a double 
boiler. Note the weight. 

2. Measure 1,000 c.c. of tap water into the sauce- 



BACTERIOLOGICAL TECHNIC 21 

pan. It is advisable to add about 200 c.c. of water to 
this to allow for evaporation. 

3. Cut and shred 15 grams of agar, add this to the 
water, bring to the boiling point, and keep at this 
temperature until the agar is completely dissolved. 
Violent boiling should be avoided and the mixture 
should be stirred, so as to prevent overheating. 

NOTE. The agar may be soaked in cold water over night. 
This removes some of the impurities and renders the agar more 
readily soluble. 

4. Add 3 grams extract of beef and 10 grams Witte's 
pep ton. 

5. Adjust the reaction. 

6. Adjust the weight. Place 1,000 grams weight 
and the weight of the saucepan on one side of the scales 
and then add enough water to make the saucepan with 
the agar balance. If the weight is too high, it should 
be boiled gently until the weight has been brought 
down to the proper amount. 

7. Make a paper filter as described below and 
arrange a retort stand as illustrated in Fig. 12. 

8. When at the boiling point filter the agar and dis- 
tribute into culture tubes. 

9. The tubed agar should be sterilized in the auto- 
clave for 5 minutes. 

For agar slants each tube should contain about 7 c.c. 
or be filled one-third of the length of the tube. For 
plating, the tube should be half filled and contain 10 
c.c. Slants are prepared by allowing the agar after 
sterilization to cool in a slanting position. If it is de- 
sired to slant a large number a whole basket may be 
put in a slanting position.. When a few tubes only 



22 LABORATORY GUIDE IN BACTERIOLOGY 



are required they may rest with the plugged end on a 
glass rod or rubber hose until the agar has solidified. 

For the purpose of filtering media heavy filter 
paper (Schleicher and Schiill No. 598) of the best 
quality only should be used. This is especially im- 
portant when filtering agar or gelatin. After the filter 



CK 



a 



FIG. 12 

Apparatus for Filtering 
Media 

a. Filter 

b. Large funnel 

c. Small funnel 

d. Rubber hose 

e. Pinchcock 
/. Pipette 

g. Culture tube 




has been folded and inserted into the funnel hot water 
should be run through the filter, until it is soaked 
and warm. 

Method of folding filters (Fig. 13). a and b) Take 
a square piece of filter paper twice as wide as the depth 
of the funnel and fold to half the size so as to make 
No. i cover No. 2. (Compare with Fig. 13.) 



BACTERIOLOGICAL TECHNIC 23 

c) Fold this to make i cover 2 and 3 cover 4 (result 
Fig. 13^). It consists of four layers and forms a square. 

d) Fold the upper part, consisting of two layers, 
from i to 2 (Fig. 13^). The shaded triangle, 2-3-4, 
now has six layers; the other, 1-3-4, two layers. 



a, 





FIG. 13 

Method of Folding Paper Filter 
(For reference letters see text) 



e) Fold the upper double layer so as to make 2 
cover a point in the diagonal at 5, taking care to make 
a sharp point at 4 (result Fig. 130). The shaded part 
is now eight layers deep. 

/) Turn the folded part face down and repeat 



24 LABORATORY GUIDE IN BACTERIOLOGY 

operations exactly on the other side as in d and e (re- 
sult Fig. i 3 /). 

g) Take up and open the large middle fold (result 
Fig- i3g). The two halves must now be symmetrical. 

h) Fold so as to make the lines 1-3 and 1-4 meet at 
the center line 1-2 (result Fig. i$h). 

i) Now pick up and fold backward so as to have 
i cover 2 in the back (Fig. 13*'). 

j) Cut through the line 1-2 and open up. The 
extreme ends will be found without a fold and may be 
folded so as to "make 9 sharp edges. 

This filter is inserted evenly into the funnel, spread- 
ing the folds at a distance from each other as nearly 
alike as possible. Care should be taken to make the 
folds and the point sharp, as this insures rapid filtration 
and prevents the filter from tearing. 

If a vacuum pump is available, the medium may be 
filtered rapidly by the use of suitable apparatus, as 
illustrated in Fig. 14. At the connection with the 
vacuum pump a valve should be inserted or a flask 
arranged as in the illustration, to prevent the water 
from entering the flask if the water pressure should be 
reduced suddenly. 

If some precautions are properly observed, chiefly 
the making of a good filter with sharp edges and a 
sharp point, and the soaking of this in hot water, there 
is no difficulty in filtering agar or gelatin successfully 
in a short time. There is some danger of the point 
of the filter breaking when the hot medium is poured 
on. This may be avoided by folding a second filter 
of about two inches diameter and fitting this small 
filter on the bottom and outside of the larger one. 



BACTERIOLOGICAL TECHNIC 25 

The basket which is to receive the tubes after filling 
should be placed in an inclined position, as this facili- 
tates the proper arrangement of the tubes. In filling 
the tubes the pipette at the end of the rubber hose 




FIG. 14 
Filtering Media by Means of Vacuum Pump 

a. Liquid medium e. Reflux flask 

b. Absorbent cotton /. Rubber stopper with two holes 

c. Rubber stopper g. Connection with aspirator 

d. Filtered medium 

should be inserted to a depth of at least two inches 
and when the proper amount has been discharged 
should be removed carefully so as to avoid wetting 
the mouth of the tube. A wet tube-mouth will cause 



26 LABORATORY GUIDE IN BACTERIOLOGY 

the cotton plug to stick to the glass, and later not only 
occasion much annoyance to the person using the tube 
but expose the medium in it to danger of contamina- 
tion. 

EXERCISE 3. PREPARATION OF DEXTROSE AGAR 

Dextrose is added to agar for the demonstration of 
gas-forming organisms. Dextrose is decomposed by 
these bacteria with gas formation, the gas appearing 
as bubbles in the medium. 

Dextrose agar is prepared by adding a definite 
amount of dextrose, usually i per cent, to filtered agar. 

NOTE. Dextrose agar cannot be distinguished from plain 
agar by appearance. It is therefore necessary either to label 
the tubes or to separate dextrose agar tubes from plain agar 
tubes in a basket by tying a piece of string or inserting a piece of 
paper between. 

EXERCISE 4. PREPARATION OF PEPTON GELATIN 

1. Weigh the saucepan and measure 1,000 c.c. of 
tap water into it. To this 200 to 300 c.c. of water 
should be added to allow for evaporation. 

2. Dissolve 3 grams extract of meat and 10 grams 
pepton. 

3. When boiling dissolve 10 per cent gelatin in 
cold weather and 12 per cent in hot weather. The 
gelatin must be of the best quality (gold label) and 
should be dissolved slowly, taking a few leaves at a 
time, and with constant stirring. 

4. When completely dissolved, adjust the reaction 
as directed in the preparation of agar. Gelatin con- 
tains an appreciable amount of acid and it will require 
more NaOH solution for neutralization than agar. 



BACTERIOLOGICAL TECHNIC 27 

5. Cool to about 60 C. Dissolve the whites of 
two or three eggs, or about 10 grams of pure powdered 
egg albumin in about 100 c.c. of tap water. Mix this 
solution with the gelatin and heat slowly to the boiling 
point, placing a piece of asbestos under the pan unless a 
double boiler is used. Boil gently until the egg white 
or egg albumin has coagulated and a solid film has 
formed which mechanically incloses the impurities. 

6. Adjust the weight to 1,000 grams, allowing for 
the weight of the pan. Filter and tube, as in the 
preparation of agar. 

7. Sterilize in the autoclave for 5-8 minutes at 
120 C., or in the arnold for three successive days. 

If sterilized in the autoclave, care should be taken 
uot to allow the temperature to go beyond i2oC., 
and gelatin should not be permitted to remain at this 
temperature beyond the prescribed time. Gelatin is 
readily decomposed by heat and then does not solidify 
after cooling. 

EXERCISE 5. PREPARATION OF LITMUS MILK 

Milk is one of the most important culture media. 
Only the cleanest milk obtainable should be used. 
"Certified milk" is most suitable. In many cases com- 
mercial milk powder may be used. If certified skim- 
med milk or fat-free milk is available step i is omitted. 

1. Separate five-sixths of the cream from the milk. 

2. Add a sufficient quantity of tincture of litmus to 
impart a decided blue color to the milk. If a solution 
of Merck's pure extract of litmus 1:100 is at hand 
about 5 per cent of this will be sufficient. 

3. Distribute in culture tubes and sterilize in the 



28 



LABORATORY GUIDE IN BACTERIOLOGY 



autoclave for 5 minutes at 120 C. After sterilization 
the blue is usually more or less lost, but returns upon 
standing. 

EXERCISE 6. PREPARATION OF POTATO 

i. Select several large potatoes, and cleanse by 
brushing the dirt off, cutting out the eyes and other 
blemishes, and washing in water. 




FIG. 15 

Potato Cylinder 

(showing diagonal for 

cutting) 



FIG. 16 FIG. 17 

Ordinary Style of Potato Tube Potato Tube 
a. Potato b. Cotton 



2. Punch out cylinders with a borer of suitable size, 
trim them, and cut each cylinder into two equal parts 
on a diagonal line (Fig. 15). 



BACTERIOLOGICAL TECHNIC 29 

3. Immerse the pieces in running water for 24 hours. 

4. Trim the pieces of potato so they slide down 
the tube and insert one half-cylinder into each potato 
tube. The wide end rests on the constriction of the 
tube. Pour a small amount of water into it, and 
fill about one-half of the part of the tube below the 
constriction. Large culture tubes without constric- 
tions may be used. A small amount of cotton should 
be pushed to the bottom of these and the cotton soaked 
with water. The potato then rests on the cotton 
(Figs. 16, 17). 

5. Sterilize in the autoclave for 8 to 10 minutes 
at 120 C., or in the arnold for three consecutive days. 

NOTE. Potatoes usually harbor a spore-bearing bacillus, the 
spores of which are highly resistant. Therefore a longer expo- 
sure iii the autoclave is necessary to insure sterilization. 

SUBSTITUTE FOR POTATO 

1. Dissolve 15 grams agar in 600 c.c. water and 
filter. 

2. Dissolve the following salts in 200 c.c. water: 

Asparagin 5 grams 

K 2 HPO 4 2 grams 

Na 2 HPO 4 2 grams 

MgSO 4 2 grams 

CaCl 2 2 grams 

Ammonium lactate 2 grams 

3. Add the solution of salts to the hot agar. 

4. Dissolve 10 grams pep ton. 

5. Adjust the reaction to the neutral point with 
phenolphthalein.. 

6. Suspend 30 grams washed starch in water. 

7. Add the starch suspension slowly with constant 
stirring to the previous mixture. 



30 LABORATORY GUIDE IN BACTERIOLOGY 



The weight of the mixture should be 1,000 grams. 
Tube hot, without filtration, through a pipette with a 
wide aperture. Autoclave for five minutes at 120 C., 
and allow to cool in a slanting position. 

EXERCISE 7. STANDARD METHOD OF PREPARING BROTH, 

NUTRIENT GELATIN, AND NUTRIENT AGAR 

STANDARD METHODS FOR THE EXAMINATION OP WATER AND 

SEWAGE. AMERICAN PUBLIC HEALTH ASSOCIATION 

755 BOYLSTON STREET, BOSTON, MASS. 



Broth 


Gelatin 


Agar 






Boil 10 or 15 g. thread agar 
in 500 c.c. water for half an 
hour and make up weight 
to 500 g v or digest for 10 
minutes in the autoclave. 
Let this cool to about 60. 


2. Infuse 500 g. lean meat 24 
hours with i ,000 c.c. of dis- 
tilled water in refrigerator. 


Ditto. 


Infuse 500 g. lean meat 
24 hours with 500 c.c. of 
distilled water in refriger- 
ator. 


3. Make up any loss by evap- 
oration. 


Ditto. 


Ditto. 


4. Strain infusion through 
cotton flannel. 


Ditto. 


Ditto. 


5. Weigh filtered infusion. 


Ditto. 


Ditto. 


6. Add i % Witte's pepton. 


Ditto. 
And 10 % gold 
label sheet gel- 
atin. 


Add 2 % Witte's pepton. 


7. Warm on water bath, stir- 
ring till pepton is dis- 
solved, and not allowing 
the temperature to rise 
above 60. 


Warm on water 
bath until pep- 
ton and gela- 
tin are dis- 
solved, not 
above 60. 


Warm on water bath until 
pepton is dissolved, not 
above 60. 


8. 




To 500 g. of meat infusion 
add 500 c.c. of the 3% 
agar, keeping the tem- 
perature below 60. 



9. Titrate after boiling one minute to expel carbon dioxid. 

10. Adjust reaction to i% acid by adding normal hydrochloric acid or sodium 
hydrate as required. 

11. Heat over boiling water (or steam) bath for 40 minutes. 
la. Restore loss by 



g water (or s 

evaporation. 



BACTERIOLOGICAL TECHNIC 31 

EXERCISE 8. PREPARATION OF BROTH FROM 
FRESH MEAT 

The following method has some advantages over 
the previous method and is specially adapted to the 
preparation of diphtheria toxin: 

1. Clean one pound beef or veal of adhering fat, 
etc., and grind in a meat chopper. 

2. Cover with one liter of water and digest over 
night at room temperature. 

3. Heat to 60 C. and digest at this temperature for 
two hours. 

4. Boil for 30 minutes. 

5. Press the liquid from the meat in a meat press 
(Fig. 1 8). Mix the meat with 

some more water, press out 
again, and bring the volume of 
the two combined liquids to I 
liter. 

6. Dissolve in this 20 grams 
pepton and 5 grams sodium 
chlorid. 

7. Adjust the reaction to 
i . 2 per cent acid with phenol- 
phthalein as indicator. 

8. Filter until perfectly clear 
and sterilize in the autoclave. 

FIG. Tg 
Meat Press 

EXERCISE 9. PREPARATION OF BLOOD SERUM 

i. Fresh ox blood, horse blood, or dog's blood, 
collected in sterile containers, is set aside in an ice 
chest until the serum has separated. 




32 LABORATORY GUIDE IN BACTERIOLOGY 

2. Three parts of serum are mixed with i part 
broth containing i . 5 per cent dextrose. 

3. The mixture is filtered and tubed. 

4. Place in a Koch inspissator (Fig. n). 

5. Incline the inspissator to the proper angle, so as 
to produce a large sloping surface of the serum. 

6. Heat slowly to the boiling point and boil for 5 
minutes. This process is to be repeated on three or 
four successive days. 

NOTE. 25 c.c. water should be placed in the inspissator with 
the tubes, so as to keep the air in the inspissator saturated. 
Blood serum may be sterilized in a shorter time by the use of 
the autoclave (see p. 16). 

EXERCISE 10. PREPARATION OF BEERWORT MEDIA 

Hopped beerwort may be obtained from a brewery. 
Beerwort media are used chiefly for the cultivation of 
yeasts and molds, these preferring media of the acidity 
of beerwort. Beerwort should be autoclaved, cooled, 
and filtered before tubing, otherwise a precipitate will 
form in the tubes. 

Liquid beerwort. Place about 7 c.c. of beerwort in 
culture tubes and sterilize at 120 C. for five minutes. 

Beerwort gelatin. Dissolve 10 to 12 per cent gold 
label gelatin in beerwort and sterilize at 120 C. in the 
autoclave. 

Beerwort agar. Dissolve i . 5 per cent agar in beer- 
wort and sterilize at 120 C. in the autoclave. 

EXERCISE II. PREPARATION OF YEAST- WATER MEDIA 

Yeast water. One liter of washed yeast or one 
pound of pressed yeast is boiled in two liters of water 
for one hour. The reaction is made neutral to phenol- 



BACTERIOLOGICAL TECHNIC 33 

phthalein, the solution is then filtered and sterilized 
in the arnold for three successive days. 

Dextrose yeast water. Dissolve 10 to 15 per cent 
dextrose in yeast water without adjusting the reaction. 



EXERCISE 12. PREPARATION OF MEDIA FOR THE DIF- 
FERENTIATION OF B. coli AND B. typhosus 
MacConkey's bile-salt agar 

Nutrient agar 100 c.c. 

Sodium taurocholate 0.5 per cent 

Pepton 2 per cent 

Boil and filter, add 2 per cent lactose, tube and steril- 
ize in the arnold. 

MacConkey's bile-salt broth 

Sodium taurocholate 0.5 per cent 

Pepton 2 per cent 

Dextrose 0.5 per cent 

Dissolve in beef broth by boiling, filter, and add 
litmus solution. Sterilize in the arnold. 
Aesculin bile-salt agar 

Agar 15 g rams 

Commercial bile-salt 2.5 grams 

Pepton (Witte) 10 grams 

Distilled water 1,000 c.c. 

Boil until dissolved and neutralize with n.NaOH. 
Cool to 60 C., add the whites of two eggs or a solu- 
tion of egg albumin, bring to boiling point, and filter 
when the albumin has coagulated. Neutralize again, 
if necessary, and add to the hot filtrate i gram aesculin 
and i gram iron citrate in scales. The final reaction 
should be 0.6 per cent acid. 



34 LABORATORY GUIDE IN BACTERIOLOGY 

Drigalski and Conradi's medium (modified by 
Harris) 

Dextrose-free broth 2,000 c.c. 

Nutrose 20 grams 

Agar 40 grams 

Boil, dissolve, neutralize to phenolphthalem, auto- 
clave at 120 C. for 5 minutes. Clarify with whites of 
four eggs (or powdered egg albumin), and filter. Then 
add after dissolving separately in water: 

Lactose 30 grams 

Litmus solution (see p. 35} 260 c.c. 

Crystal violet (o.i per cent solution) 20 c.c. 

Tube and sterilize in the arnold. 
Parietti's solution 

Carbolic acid 5 c.c. 

Hydrochloric acid 4 c.c. 

Water 100 c.c. 

Phenol media. One part carbolic acid (phenol) is 
added to 1,000 parts medium. 

Hiss's plating medium for colon-typhoid differ- 
entiation 

Agar 15 grams 

Gelatin 15 grams 

Extract of meat 5 grams 

NaCl 5 grams 

Dextrose 10 grams 

Distilled water 1,000 c.c. 

Hiss's tube medium 

Agar 5 grams 

Gelatin 80 grams 

Extract of meat 5 grams 

NaCl 5 grams 

Dextrose 10 grams 

Distilled water 1,000 c.c. 



BACTERIOLOGICAL TECHNIC 35 

Hesse's medium 

Agar 5 grams 

Pepton 10 grams 

Extract of meat * . 5 grams 

NaCl 8.5 grams 

Distilled water 1,000 c.c. 

Capaldi's medium 

Pepton 20 grams 

Gelatin 10 grams 

Agar 20 grams 

Dextrose or mannit 10 grams 

NaCl 5 grams 

KC1 5 grams 

Distilled water 1,000 c.c. 

Malachite green media. Two to 3 c.c. of a 2 per 
cent solution of "Hochst 120" malachite green solu- 
tion is added to broth (alkaline to litmus). 

Ox-bile medium. This culture medium is used for 
the detection of B. coli in water. Dissolve i per cent 
lactose and i per cent pepton in fresh ox bile, filter and 
fill in fermentation tubes and sterilize in the arnold for 
three successive days. 

EXERCISE 13. PREPARATION OF MEDIA FOR WATER 
AND MILK EXAMINATION 

Litmus solution. Dissolve i part Merck's pure 
extract of litmus in 100 parts of water, filter, and 
sterilize in the autoclave for 5 minutes at 120 C. 

Litmus lactose agar and litmus dextrose agar. 
Dissolve i per cent lactose or dextrose in sugar-free 
agar. The litmus should be added in the petri dish 
before use from a sterile tube of litmus solution by 
means of a sterile i c.c. pipette. 



36 LABORATORY GUIDE IN BACTERIOLOGY 

Litmus lactose gelatin and litmus dextrose gela- 
tin. Dissolve i per cent lactose or dextrose in sugar- 
free gelatin, containing 13 per cent gelatin. The tubes 
should contain 8*c.c. of the medium. After adding 
i c.c. litmus solution and i c.c. of the material to be 
plated the medium will contain 10 per cent gelatin 
and remain solid. 

Mannit agar. Dissolve i per cent mannit in sugar- 
free agar. 

EXERCISE 14. PREPARATION OF WHEY MEDIA 

Litmus whey (Petruschky, modified by Durham). 
Casein is precipitated from milk with rennet extract. 
The whey is neutralized with 4 per cent citric acid 
solution and heated on the water bath for half an hour. 
It is then filtered, and litmus solution added until a 
decided blue color is obtained. Sterilize in the auto- 
clave. 

Whey gelatin. Add 10 per cent gelatin to clarified 
whey. 

Whey agar. Add a few drops acetic acid to boiling 
milk until the casein is precipitated. Neutralize, or 
bring to a reaction of i per cent acid if desired, and 
dissolve i per cent pepton, 2 per cent dextrose, and i . 5 
per cent agar. Filter, tube, and sterilize in the 
autoclave. 

EXERCISE 15. PREPARATION OF GLYCERIN MEDIA 

Glycerin broth. Add 6 per cent pure glycerin to 
ordinary broth. 

Glycerin agar. Add 6 per cent pure glycerin to 
nutrient agar. 



BACTERIOLOGICAL TECHNIC 37 

Glycerin egg medium. Add 6 per cent glycerin to 
the egg mixture before heating. 

Glycerinated potato. Prepare potatoes in the usual 
manner and soak for 24 hours in a 25 per cent glycerin 
solution in distilled water. 

Substitute for glycerinated potato. Mix 6 per cent 
glycerin with the substitute for potato described on p. 
29. 

EXERCISE 1 6. PREPARATION OF EGG MEDIA 

Dorset's egg medium. Eggs are broken into a 
flask and the yolks broken with a platinum needle or 
glass rod. The flask is gently shaken until the yolks 
and whites are thoroughly mixed. Foam formation 
should be avoided. Distribute in culture tubes and 
sterilize in a Koch inspissator or autoclave in the same 
manner as blood serum. 

Dorset's egg-yolk medium. Add 5 to 10 c.c. of 
sterile distilled water to the yolks of three or four eggs, 
and treat the same as in the above egg medium. 

Capaldi's egg medium. A few loopfuls of egg yolk 
are added to a tube of liquefied agar, previously cooled 
to 45 to 47 C. 



EXERCISE 17. MEDIA FOR THE STUDY OF SOIL 
BACTERIA 

Winogradsky's solution 

MgSO 4 o . 50 gram 

CaCla o.oi gram 

NaCl 2 . oo grams 

Distilled water 1,000 c.c. 



38 LABORATORY GUIDE IN BACTERIOLOGY 

Solution for nitrite formation 

FeSO 4 0.4 gram 

MgSO 4 0.5 gram 

K 2 HPO 4 i gram 

NaCl . 2 grams 

(NH4) 2 SO 4 i gram 

Distilled water 1,000 c.c. 

Dissolve 20 grams agar in the above solution. 
After solution add 10 grams precipitated CaCO 3 . 
Shake and tube. Sterilize in the autoclave. 

Preparation of silica jelly. Mix 100 c.c. HC1, 
specific gravity i . 10 Beaume's scale at 60 F., with 
100 c.c. sodium silicate, specific gravity i .09 Beaume's 
scale at 60 F. Place in collodion sacs and dialyze in 
running water for 12 hours. Prepare the following 
solution: 

(NH 4 ) 2 SO 4 0.40 gram 

MgSO 4 o . 05 gram 

KH 3 PO 4 o . 10 gram 

Na 2 CO 3 o . 90 gram 

CaCla o.oi gram 

Dissolve these salts in the smallest amount of dis- 
tilled water possible. Heat both the salt solution and 
the silica jelly to boiling, cool rapidly without stirring. 
Mix and pour on petri dishes or tubes and sterilize in 
the autoclave at 110 C. The tubes should be placed 
in a slanting position. 

Synthetic agar for plating 

Dextrose 10 grams 

MgSO 4 0.2 gram 

K 3 HPO 4 0.5 gram 

Pepton o . 05 gram 

Agar 20 grams 

Distilled water 1,000 c.c. 

Dissolve, filter, tube, and sterilize in the autoclave. 



BACTERIOLOGICAL TECHNIC 39 

Solution for testing the formation of nitrates 

MgSO 4 0.3 gram 

FeS0 4 0.4 gram 

NaCl 0.5 gram 

K 2 HPO 4 0.5 gram 

Fused Na 2 CO 3 i gram 

NaNO 2 i gram 

Distilled water 1,000 c.c. 

Dissolve 20 grams agar in the above solution. 
Sterilize in the autoclave. The salt solution may also 
be added to the silica jelly. For plating fill in culture 
tubes. 

Solution for testing the assimilation of atmospheric 
nitrogen 

i. Solution 

KH 2 PO 4 i gram 

MgSO 4 o. 50 gram 

NaCl o . 01 gram 

FeSO 4 o . 01 gram 

MnSO 4 o . 01 gram 

Dextrose 20 grams 

Distilled water 1,000 c.c. 

Dissolve 20 grams agar in this solution if intended 
for plating and tubing. Sterilize in the autoclave. 

2. Mannit Solution 

K 2 HPO 4 o . 20 gram 

CaCl 2 0.02 gram 

MgSO 4 o . 20 gram 

Fe 2 Cl6 o . 01 gram 

Mannit 15 grams 

NaOH solution until neutral to phenolphthalem 
Distilled water 1,000 c.c. 

Dissolve 20 grams agar in this solution if intended 
for plating and tubing. Sterilize in the autoclave. 



40 LABORATORY GUIDE IN BACTERIOLOGY 

Solution for testing the denitrification of nitrates 
to free nitrogen (Giltay solution) 

Solution i 

Dextrose 10 grams 

Distilled water 250 c.c. 

Solution 2 

KNOj i . oo gram 

MgSO 4 2 . oo grams 

Citric acid 5 . oo grams 

KH 2 PO 4 2 . oo grams 

CaCla o . 2 gram 

Fe 2 CU o . 01 gram 

Distilled water 250 c.c. 

Make solution i neutral to phenolphthalei'n with 

10 per cent NaOH or KOH solution. Mix solutions 

i and 2 and make up to 1,000 c.c. Dissolve 20 grams 

agar in the solution, tube, and sterilize in the autoclave. 

Synthetic agar for quantitative examination of soil 

Dextrose 10 grams 

MgSO 4 0.2 gram 

K 2 HPO 4 0.5 gram 

Pepton o . 05 gram 

Agar 20 grams 

Distilled water 1,000 c.c. 

Pepton solution for ammonification 

Distilled water 1,000 c.c. 

Pepton 10 grams 

Dissolve and sterilize in the autoclave. 

EXERCISE 1 8. PREPARATION OF MISCELLANEOUS 
MEDIA 

Blood agar. Fresh blood obtained under aseptic 
precautions is smeared over the surface of agar, or 



BACTERIOLOGICAL TECHNIC 41 

blood may be mixed with agar, previously liquefied 
and cooled to 50, in various proportions. The agar 
should contain 2 to 3 per cent agar according to the 
amount of blood to be added. 

Bread-paste medium. Bread is cut into slices, 
dried in an oven, pulverized, and distributed in 100 c.c. 
flasks until the layer on the bottom of the flask is about 
half an inch thick. Water is added gradually until 
the surface of the bread is moist. Sterilize in the 
arnold. 

Hay infusion. 10 grams of chopped hay are 
macerated in 1,000 c.c. water in the water bath for 
three hours. Filter and sterilize in the autoclave. 

Wine must. Wine must is diluted with four times 
its volume of water. Dissolve o . 5 per cent ammonium 
tartrate, macerate in the water bath for i hour, filter 
and sterilize in the arnold for three successive days. 

Acid broth. Add 0.5 per cent acetic acid to 
ordinary broth. 

Calcium carbonate broth. A few lumps of marble 
added to ordinary broth. 

NITRATE MEDIA 

Nitrate broth. Add 5 parts potassium nitrate to 
each liter of ordinary broth. 

Nitrate, solution. 5 c.c. of a 2 per cent aqueous 
potassium nitrate solution are added to a solution of 
i gram pepton and i gram dextrose in 1,000 c.c. water. 



42 LABORATORY GUIDE IN BACTERIOLOGY 

EXERCISE 19. PREPARATION OF NON-PROTEIN MEDIA 
(SYNTHETIC MEDIA) 

Jordan's non-protein medium 

Redistilled water 1,000 c.c. 

Asparagin 2 grams 

MgSO 4 i gram 

K2HPO 4 i gram 

Dissolve and sterilize in the autoclave. 

Uschinsky's medium (FrankeFs modification) 

Water 1,000 c.c. 

Asparagin 4 grams 

Ammonium lactate 6 grams 

Na 2 HPO 4 2 grams 

NaCl 5 grams 

Dissolve and sterilize in the autoclave. 

Raulin's solution for the cultivation of molds 

Distilled water 1,500 c.c. 

Cane sugar 70 grams 

Tartaric acid 4 grams 

Ammonium nitrate 4 grams 

Potassium carbonate 0.6 gram 

Ammonium phosphate 0.6 gram 

Magnesium carbonate 0.4 gram 

Ammonium sulphate o. 25 gram 

Zinc sulphate 0.07 gram 

Iron sulphate 0.07 gram 

Potassium silicate o . 07 gram 

NOTE. Culture media should be stored in a dark, cool place. 
If they are to be kept for a considerable length of time, the tubes 
should be sealed with paraffin or with rubber caps. They should 
be protected from dampness, as mold fungi are apt to alight on 
the cotton stoppers and send their filaments into the tubes. 
Before inoculation each tube should be examined, and those 
which are cloudy or contain colonies of bacteria or molds should 



BACTERIOLOGICAL TECHNIC 43 

be rejected. If the medium has shrunk from the effects of evapo- 
ration it should be rejected, or the evaporated water may be re- 
placed and the medium sterilized again. 

SECTION 6 
PREPARATION OF STAINING SOLUTIONS 

Saturated alcoholic solutions of stains are prepared 
by covering the anilin dye with absolute or 96 per 
cent alcohol and allowing it to stand with frequent 
agitation until no more stain is dissolved. Other 
solutions for the preparation of stains are: 

1. Solution of potassium hydrate in water i : 10,000. 

2. Solution of carbolic acid in water (5 per cent). 

3. Anilin water, prepared by shaking 2 c.c. anilin 
oil with 100 c.c. water and filtering through filter 
paper until clear. 

The following four stains are most commonly in 
use for morphological studies of bacteria: 

1. Loffler's methylene blue 

Saturated alcoholic solution of methylene blue 30 c.c. 

Potassium hydrate in distilled water i : 10,000! 70 c.c. 

2. Ziehl-Neelsen's carbol-fuchsin 

Saturated alcoholic solution of fuchsin 10 c.c. 

5 per cent solution carbolic acid in distilled water 90 c.c. 

3. Ehrlich's anilin gentian violet 

Saturated alcoholic solution of gentian violet 25 c.c. 

Anilin water (2 per cent) 75 c.c. 

4. Gram's iodin solution 

lodin i gram 

Potassium iodid 2 grams 

Dissolve in a few cubic centimeters of distilled 



44 LABORATORY GUIDE IN BACTERIOLOGY 

water. After solution add enough distilled water to 
bring the volume to 300 c.c. 

Other useful stains are the following: 

Delafield's hematoxylin 

Hematoxylin crystals 4 grams 

Alcohol 25 c.c. 

Ammonia alum 50 grams 

Water 400 c.c. 

Glycerin 100 c.c. 

Methyl alcohol 100 c.c. 

Alum hematoxylin 

1. Hematoxylin 2 grams 

Absolute alcohol 100 c.c. 

2. Ammonia alum 2 grams 

Water 100 c.c. 

Mix i and 2 and add 

Glycerin 850 c.c. 

Glacial acetic acid 100 c.c. 

Allow to stand for one month before using. 

Bismarck brown 

Bismarck brown 0.5 gram 

Water ; 100 c.c. 

Safranin 

Safranin 0.5 gram 

Water 100 c.c. 

Carbolic thionin blue (Nicolle) 

Thionin blue i gram 

Carbolic acid 2.5 grams 

Water 100 c.c. 

Alum carmin 

Alum 2.5 grams 

Carmin i . o gram 

Water . . .100 c.c. 



BACTERIOLOGICAL TECHNIC 45 

Kuhne's methylene blue 

Methylene blue 1.5 grams 

Absolute alcohol 10 c.c. 

Carbolic acid solution (5 per cent) . . 100 c.c. 

Carbolic gentian violet (Nicolle) 

Gentian violet (sat. alcoh. sol.) 10 c.c. 

Carbolic acid i gram 

Water 90 c.c. 

Pappenheim's Stain 

Sat. aqueous solution of methyl green 3-4 parts 
Sat. aqueous solution of pyronin i-i . 5 parts 

Mix previous to staining. Apply for 20 seconds. 
The mixture is dependable for a few weeks only. 

SECTION 7 

THE MICROSCOPE 

(Fig. 19) 

The compound microscope is a necessary adjunct 
to any kind of bacteriological work. For this work 
three objectives (Leitz No. 3, No. 6, or No. 7 and T \ oil 
immersion, Zeiss No. A or No. AA, No. D or No. DD, 
and 2 mm. oil immersion) and two oculars (Nos. 2 and 
4) are indispensable. For the intelligent manipula- 
tion of the microscope it is useful to understand the 
underlying optical principles, which may be studied 
from special works on the subject. 

References 

S. H. Gage, The Microscope. 

Carpenter and Dallinger, The Microscope and Its Revelations. 

For use in the laboratory it will be sufficient to call 
attention to some of the most important points to be 
observed. 



46 LABORATORY GUIDE IN BACTERIOLOGY 

The usual pattern of microscope consists of two 
main parts: the stand, and the optical parts (Fig. 19) 
which are attached to the stand. 

The stand consists of a body tube, draw tube, 
coarse adjustment, fine adjustment (micrometer screw) 
in a pillar, nose-piece, stage with clips for holding the 
object, main pillar, and the horseshoe base. At the 
junction of the main pillar and the fine-adjustment 
pillar is the inclination joint. 

The draw tube, regulating the focal length, which 
varies in different instruments, should be raised to 16 
or 17 mm. If a nose-piece is attached, the width of 
this must be deducted from the tube length. 

The optical parts are the oculars, the objectives, the 
substage condenser, and the mirror. The ocular is a 
combination of lenses, which slips into the top of the 
draw tube and is nearest the eye. The objective is a 
combination of lenses which is screwed into the nose- 
piece and fits to the lower end of the draw tube. The 
substage condenser fits under the stage. It concen- 
trates the light on the object and is raised for high 
powers or lowered for low powers. At the lower end 
of this condenser is the iris diaphragm, which is regu- 
lated by a small lever with a milled head, and serves 
the purpose of regulating the light supply. The mirror 
has two sides, a concave and a flat one. 

In the manipulation of the compound microscope 
the following points should be observed: 

1. Keep the instrument clean. When not in use, 
lock it in the case or cover it with a bell-jar. 

2. When carrying the instrument, grasp it by the 
main pillar underneath the .stage, not by the fine- 




-..e 



a. Ocular 



FIG. 19 
Microscope (after E. Leitz) 

Iris diaphragm and condenser 



b. Place where virtual picture is formed k. Objective 



c. Body tube 

d. Coarse adjustment 

e. Micrometer screw 
/. Inclination joint 
g. Horseshoe base 

ft. Mirror 



/. Nose-piece 

m-o. Picture as it appears to the eye 

p-q. Object (cover slip) 

r . Adjustment spring 

s. Stage 

/. Object (slide) 



^ 

* 1 

*} ^rfl"* rt 

** rt n 1 * ""i '</*** 

48 LABORATORY GUIDE IN BACTERIOLOGY 

adjustment pillar. The fine adjustment consists of 
a delicate screw-thread, which is easily damaged. 

3. The lenses, condenser, and mirror, when necessary, 
should be wiped with Japanese lens paper, never with 
any coarse material. 

4. For cleaning use a damp cloth. For wiping the 
lenses use water or xylol. Never use alcohol, as this 
dissolves the cement holding the lenses in place and 
also injures the lacquer. 

5. Do not take the instrument apart. The working 
parts are of delicate nature and easily injured. Be 
careful not to drop the oculars or objectives. 

6. The inclination joint can be used only with dry 
lenses and dry objects, not with the oil-immersion or 
hanging-drop preparations. 

7. After placing the object on the stage, focus with 
a low power and the coarse adjustment. With high 
powers use the coarse adjustment first and the fine 
adjustment afterward. The free use of the fine adjust- 
ment saves the accommodation of the eye. As the 
eye is capable of accommodating itself to distances, it 
may, with an effort, distinguish a picture which is not 
in perfect focus. This effort is saved by using the 
fine adjustment. 

8. Raise the draw tube by means of the coarse 
adjustment before changing the objectives or examin- 
ing a different preparation. 

9. Before focusing, obtain as good light as possible 
by turning the mirror, and then regulate the supply 
by the diaphragm. Always use reflected light, never 
direct sunlight. 

10. When focusing an object, lower the draw tube 
until the lens almost touches the cover glass. This 



BACTERIOLOGICAL TECHNIC 49 

can be seen by looking at the instrument from one side 
and watching the reflection of the objective in the cover 
glass. Then, with the eye at the ocular, slowly focus 
up. Do not focus down with the eye at the ocular, as 
the lens may then come into violent contact with the 
object, destroying the latter and injuring the lens. 

11. For living or transparent objects use as little 
light as possible. For stained or opaque objects more 
light is necessary. 

12. Do not use higher powers than are necessary. 

13. To use the oil-immersion lens, place a drop of 
clear cedar oil, free from dust and air bubbles, on the 
cover glass, which must be perfectly dry. In this case, 
by careful manipulation, the objective, after being 
brought in touch with the oil by means of the coarse 
adjustment, may be gradually lowered by the fine 
adjustment until the object is focused; or better, 
lower the objective until almost in touch with the cover 
glass, and focus up. High powers require the use of 
a homogeneous liquid between the cover glass and the 
front lens of the objective, to avoid loss of light by 
refraction. As a bundle of rays disperses when enter- 
ing a thinner medium from a denser one, there is not 
sufficient light entering the objective to make objects 
discernible, when using high powers without oil. By 
the insertion of a liquid (inspissated oil of cedar) of 
nearly the same refractive index as glass, a homogene- 
ous connection is established between the cover glass 
and the objective, thus avoiding loss of light and allow- 
ing a bundle of light of sufficient power to enter the 
objective. 1 

1 For detailed description and diagrams see S. H. Gage, The 
Microscope. 



50 LABORATORY GUIDE IN BACTERIOLOGY 

14. After using the oil-immersion lens, wipe the oil 
off with lens paper. If the oil sticks to the lens, wipe 
it off with xylol, never with alcohol. At the same time 
wipe the oil off the cover slip. 

15. The microscope should stand on a firm table, 
to avoid being shaken. The table should be low enough 
so as not to necessitate bending the body. 

16. Always keep both eyes open. This saves the 
eyesight. Beginners find this a difficult rule to apply, 
but with very little practice and persistence it is 
easily accomplished. Also use both eyes alternately. 

17. It is well to move the object while bringing 
it into focus. It is then easy to feel when the lens 
touches the glass, and a moving object is seen more 
readily than a stationary one. 

1 8. Use the plane mirror in combination with the 
condenser, and the concave mirror without the con- 
denser or with artificial light. 

19. In preparing stained preparations, it may hap- 
pen that a small amount of stain remains on the upper 
side of the cover slip. Care must be taken to focus for 
a plane below this. 

SECTION 8 
SCHEME FOR ROUTINE STUDY OF BACTERIA 

This scheme is to be followed strictly in the study of 
all organisms, except when special instructions are 
given, and the student should familiarize himself with 
the different steps. 

i. The inoculation of one slant agar tube from each 
rulture supplied. These inoculated agar tubes are to 



BACTERIOLOGICAL TECHNIC $1 

be incubated at 37 for 24 hours, unless otherwise in- 
structed. 

2. At the end of 24 hours make from each agar tube: 

a) A physical description of the culture (see Section 

9)- 

b) A hanging-drop examination. 

c) A Gram stained preparation. 

d) An ordinary stained preparation. 

All stained preparations and all Gram stains should 
be preserved. 

3. Transfer from agar culture to the following 
media: 

Dextrose agar. 
Gelatin. 
Potato. 
Broth. 

Litmus milk. 

Dunham's solution, if a test for indol is to be 
made. 

These transfers, excepting gelatin, are to be placed 
in the thermostat, unless otherwise ordered. 

4. An accurate description and sketches are to be 
made of each culture (see Section 9). These descrip- 
tions should be made complete after 24 hours, and any 
changes should be noted after 48 hours and after 6 
days. (See culture charts.) 

5. Plates are to be made in agar from a 24~48-hour- 
old broth culture of each culture, unless otherwise 
directed. These plates are to be described once after 
24-48 hours. (See Section 9.) 

NOTE. The thermostat or incubator (Fig. 20) is a box 
made of copper and having double walls, between which water 



$2 LABORATORY GUIDE IN BACTERIOLOGY 

circulates. The outer surface is usually covered with asbestos 
or linoleum, so as to hold the heat. The thermostat is provided 
with two doors, the inner one of glass so as to enable the observer 
to look inside without causing a drop in temperature by admitting 




FIG. 20 
Thermostat or Incubator 

the cooler air. The outer door is covered with asbestos like 
the walls. A thermometer reaches through the upper part, 
provided with several air holes which permit free circulation 
of air. The gas supply is shut off automatically, if it should 
accidentally become extinguished. The necessary heat may also 



BACTERIOLOGICAL TECHNIC 53 

be obtained successfully by electric devices. For class use large 
incubators are constructed on similar principles, with a number 
of separate compartments for the accommodation of large num- 
bers of students. 



SECTION 9 
METHOD OF DESCRIBING CULTURES 

The following method of describing cultures should 
be carefully studied, and each suggestion should be 
considered in the description. It is of prime impor- 
tance that cultures should be closely observed, in all 
media, and accurately described and sketched, as this 
is the only method which furnishes the proper means 
of studying and determining the different species of 
bacteria. By keeping this scheme in sight while mak- 
ing a description, it will be possible after some practice 
to make perfect descriptions without its aid. After 
growth has taken place the culture should be compared 
with a sterile tube of the same batch of culture medium. 

I. Morphological characters of bacterium. 
Size. 

Facility and mode of staining. 
Gram stain. 

Special staining qualities. 
Motility. 

Present or absent. 

Sluggish or active movement. 
Flagella present or absent. 
Capsules present or absent. 
Spores present or absent. 
Involution forms. 



54 LABORATORY GUIDE IN BACTERIOLOGY 

Make a sketch of part of a field under the 
microscope. 

II. Plate cultures. 

1. Naked-eye appearance. 

a) Surface colonies. 

If colonies are too small to make observations with 
the naked eye, this fact should be stated. 
Color by reflected light. 
Approximate size. 
Elevation or depression. 
Translucency. 
Moist appearance. 
Smoothness. 
Luster. 

Liquefaction (in gelatin plates only). 
Consistency: soft, viscid, hard, chalky. 

b) Deep colonies. 
Color. 
Shape. 

2. Microscopic appearance under low power 
(No. 3 objective, Leitz). Use little light; 
the diaphragm should be almost closed, 
a) Surface colonies. 

Shape. 
Color. 

Translucency. 

Thickness (in center and edges). 
Nucleation. 
Striation. 

Granulation (if present, whether coarse 
or fine) or Homogeneity. 



BACTERIOLOGICAL TECHNIC 55 

Edge: entire or smooth, wavy, with 
pointed protuberances, serrate, den- 
tate, lacerate, fringed, hairlike 
shoots, curled, tubercle-like appear- 
ance (tuberculated). 
b) Deep colonies. 

Shape: punctiform, lanceolate, oval, 
circular, spindle-shaped, conglom- 
erate, irregular, branched, filamen- 
tous, rosette-shaped. 

Color. 

Translucency. 

Granulation. 

III. Agar slant culture. 

Limitation: confinement to needle track or 

spreading. If spreading, in what shape? 
Vigor: luxuriant, fair, or scant. 
Color: by reflected light. 
Elevation or depression, more pronounced at 

edges or in center. 
Translucency. 
Moistness. 
Smoothness. 
Luster. 

Coloration of medium. 
Odor. 

Gas formation: in culture or in medium. 
IV. Stab culture in plain agar. 

i. Surface growth (describe like agar slant). 



56 LABORATORY GUIDE IN BACTERIOLOGY 

2. Stab growth. 
Vigor. 
Extent. 
Color. 

Granulation. 
Outgrowths. 
Coloration of medium. 
Cloudiness. 
Gas formation. 

V. Stab culture in dextrose agar. 

Describe like plain agar stab and in addition 
always note presence or absence of gas 
formation. 

VI. Stab culture in gelatin. 

Describe like agar stab, and in addition 
always note presence or absence of lique- 
faction. If liquefaction is present, it may 
be saucer-shaped, turnip-shaped, conical, 
funnel-shaped, horizontal (extending the 
whole diameter of tube), sack-shaped. 
Cloudiness and presence of sediment in 
liquefied area, and color and shape of sedi- 
ment, should be described. 

VII. Potato culture. 

Describe like agar slant, adding to it the 
eventual discoloration of the medium, and 
presence of gas bubbles. 

VIII. Litmus milk culture. 

Reaction (acid, alkaline, or neutral, as indi- 
cated by color). , fc 



BACTERIOLOGICAL TECHNIC 57 

Coagulation: present or absent. 

NOTE. If acid has formed but no coagulation is evident after 
six days, heat gently and see whether coagulation then takes 
place. 

Whey: if present, clear or turbid. 

Liquefaction of coagulum (proteolysis). 

Gas formation. 

Decoloration of litmus. 

Color of cream ring. 

Odor. 

In milk the presence or absence of coagula- 
tion and proteolysis should be noted. Com- 
pare your culture with a sterile milk tube. 

IX. Broth culture. 

Cloudiness: degree and uniformity; scum 

(ring- or island-shaped). 
Precipitate, observed by shaking. Amount, 
color, formation, diffusibility, viscidity, 
amount of precipitate. 
X. Solidified blood serum. 

Describe like agar slant, and note presence or 

absence oi liquefaction. 
XI. Fermentation tubes. 

Gas formation in closed arm, percentage and 
relation of carbon dioxid to hydrogen ex- 
pressed in simple figures by the formula 
H 

C(V 
Growth in both arms or in one arm only, 

observed by cloudiness. 
Reaction: acid or alkaline to litmus. 



$8 LABORATORY GUIDE IN BACTERIOLOGY 

SECTION 10 

DIRECTIONS FOR FILLING OUT CULTURE CHARTS 

(See chart on pp. 59-60.) 
First page 

I. Name the group and organism. 
II. Indicate the source and habitat from books 
and references. 

III. Name the most important references, and 
read them. 

IV. Morphological characters. 

1. Describe the morphology opposite the 
medium from which obtained as observed 
from the stained preparation. 

2. Size: approximate estimate in microns. 
Note whether large or small, thick or 
slender, round or square ends, etc. 

3. Arrangement of bacteria: in groups, 
chains, bunches, pairs (diplococcus), sar- 
cina form, filaments, branching, etc. Also 
note different arrangements, if observed, 
in different media. 

4. Staining powers: Mark + for positive, 
for negative stains. Special stains 
must be described more fully. 

5. Motility: If absent, mark ; if present, 
-f-. In the latter case describe the char- 
acter of the movement. 

6. Spores: Absence noted by ; presence, by 
+. If present, mention the method of 
spore stain applied.. 



FROST'S CULTURE CHART (MODIFIED) 



Group 

Name of organism. 

Source 

Habitat 

References 



MORPHOLOGICAL CHARACTERS 




r 


1 Incubation 
temp. (C.) 


SKETCHES 


i. 

2. 

3- 

4- 

5- 

6. 

7- 


Form: ROD: 


Coccus: 


SPIRILLUM: 






































Size 




























































b Loffler's methylene blue 










c Gram stain 










d. Special stains, such as: (i) 
(3) "Tubercle" (4) Ca 
Motility: (a) Flagellar 


Flagella...(2)Spore... 




.... 


. (b) Molecular 




.... 






















capsules, vacuoles, granules, 
pleomorphic and involution f 










orms etc 

















PHYSIOLOGICAL CHARACTERS 

1 Relation to temperature: (a) Optimum. . . (b) Minimum. . . (c) Maximum. 

2 Relation to free oxygen: 

Relation to other agents such as: 

desiccation, light, disinfectants, etc: 

4 Pigment production: 

5 Growth in carbohydrate media: 

a. Stab or shake culture (dextrose agar): gas production 

6. Fermentation tube: (i) growth in bulb:. . (2) growth in closed arm: 



After 24 hours 
After 48 hours . 

Gas formula ~j 

d. Reaction (i) in bulb (2) in closed arm. 

6. Acid or alkali production (in litmus milk) 

7. Reduction of nitrates to nitrites to ammonia 

8. Indol production; 24 hours 48 hours days 

fecal odor; 24 hours 48 hours days. ....... 

9. Enzym production; (a) proteolytic (b) coagulative. . . . (c) diastatic. . 

10. Characteristic odor 

11. Agglutination 

12. Pathogenesia 



gas produced 


DEXTROSE 


LACTOSE 


SACCHAROSE 


















i . 








O 2 









CULTURE CHARACTERS OF 





Reaction 












of Medium, 
Incubation 
Temp. (C.) 


24 
Hours 


48 
Hours 


6 
Days 


Sketches 


(I) 


















Streak 












^ 






or Stab. 












/ \ 


















> \ 






(2) 










Potato, or 
Heinemann's 


















Synthetic 
Potato 












v ^ '' 






Medium. 










NS ^ </ 


\^^ 


(3) 


















Gelatin 












/v 
/ \ 




A 
/ ^ 


Stab. 












/ \ 




/ \ 


(4) 












/ \ 




/ \ 


Litmus 


















Milk. 












^ t 




^ ; 


(s) 












\^-^ 




^ ^ ^ J 


Broth 


















(Bouillon) 


















(6) 
Dextrose 


















Agar, or 


















Special 


















Medium. 












\ ) 




^ / 


(7) 

Agar plate, 
or roll 














tube. 














(a) Surface 
Colonies. 














(6) Deep 
Colonies. 








^ 




^^ 


(8) 














Gelatin 














plate, or 














roll tube. 














(a) Surface 














Colonies. 














(J) Deep 














Colonies. 








^-S 




^~/ 



This Culture Chart is used in connection with " A Laboratory Guide in Bacten- 

ology," by PAUL G. HEINEMANN, published by 
The University of Chicago Press, Chicago, Illinois 



BACTERIOLOGICAL TECHNIC 6 1 

7 . Note any peculiar appearance in the micro- 
scopical picture, especially involution 
forms and the presence or absence of 
capsules. If capsules are present, note 
the method of demonstration. 
V. Physiological characters. 

1. Relation to temperature: What is the 
optimum temperature? 

2. Relation to free oxygen: aerobe, anaerobe, 
facultative aerobe, or anaerobe. 

3. Relation to disinfectants, light, desicca- 
tion, heat (thermal death-point). 

4. Pigment production: If present, +; if 
absent, . In the former case, note the 
color, diffusibility, solubility, influence of 
acids and alkalis. 

5. Gas production in glucose media: to be 
filled out only in case of actual observation. 
In fermentation tubes note the growth in 
either arm or both arms, recognized by tur- 
bidity. Note the total percentage of the 
gas formed in 24 and 48 hours. Reaction 
may be tested by the addition of litmus 

TT 

solution. Gas formula expressed: T^T- 

CU 2 

6. Acid or alkali production in litmus milk. 

7. The production of indol or nitrites, or 
both, is tested on the sixth day of observa- 
tion in a culture in Dunham's pepton 
solution or sugar-free broth. 

NOTE. Indol is a decomposition product of proteins and 
belongs to the aromatic series. Nitrites result from reduction of 



62 LABORATORY GUIDE IN BACTERIOLOGY 

nitrates, or from oxidation of ammonia. The ability of organisms 
to produce these reactions is of great importance in their differ- 
entiation. A control test with a tube of sterile medium should 
be made. 

8. Enzym production: Proteolytic enzym 
production noted by the liquefaction of 
gelatin or casein. Coagulative, recognized 
by precipitation of casein, if acid forma- 
tion is absent, or present only in quantities 
less than 0.4 per cent. Amylolytic, by 
the digestion of starch (potato). 

9. Characteristic odor. 

10. Pathogenesis: What pathogenic effect has 
the organism on man? What effect on 
animals, and which animals? What dis- 
eases are caused by the organism in man 
or animal? 

NOTE. The terms "proteolysis," "enzym production," and 
"coagulation" are frequently confusing to the beginner. The 
following brief explanation will aid in an intelligent interpretation 
of the reactions observed. 

"Proteolysis" is the breaking up of complex nitrogenous 
compounds (proteins), rendering them soluble. This process is 
also expressed by the terms "peptonization" and "liquefaction." 
The liquefaction of gelatin is one kind of proteolysis. Gelatin 
is composed of nitrogenous matter (albuminoid or gelatinoid), 
and it is for this reason mainly that gelatin stab cultures are 
made. If the gelatin is liquefied, the assumption is that the 
organism is capable of producing a "proteolytic" or gelatinolytic 
enzym. In milk the process is more complex, and this medium, 
on account of its composition (fat, milk sugar, casein, lactalbu- 
min), offers excellent opportunities for the organism to develop 
different characteristics. Milk is one of the most important of 
media. The casein, contained in milk in colloid solution, may be 
precipitated by an enzym or by an acid. This precipitate forms 
the coagulum. At least 0.4 per cent of acid, which is largely 



BACTERIOLOGICAL TECHNIC 63 

lactic acid produced by splitting of milk sugar (lactose), is 
required for precipitation, and this amount of acid will turn the 
blue litmus to a decided red. A coagulum may also be produced 
by the presence of a " coagulative " or " rennet "-like enzym, 
which is the result of the metabolic activity of the organism. 
Such coagulation may take place in milk of amphoteric or alkaline 
reaction, as well as in milk of slightly acid reaction. The coagu- 
lum formed by any of the mentioned agents may gradually 
contract, and a straw-yellow, opalescent liquid will be squeezed 
out, called "whey." If the organism also produces a proteolytic 
enzym, this will attack the coagulum and gradually dissolve it 
(proteolysis, peptonization). At first the coagulum shows a 
broken-up edge; lumps separate and settle to the bottom, and 
finally the coagulum may disappear completely. Theoretically, 
coagulation is always necessary before proteolysis sets in, but in 
the case of some organisms the proteolytic enzym is so powerful 
as to produce immediate solution of the casein. (See Figs. 21, 
22, 23, 24.) 

Another phenomenon frequently observed in litmus milk 
is the decolorization of litmus, whether this be pink or blue. 
This is due to the fact that the organism takes up the oxygen 
necessary to maintain the coloration. It may be frequently 
observed that at the surface, where atmospheric oxygen has 
access, the color remains or is restored. The color may also be 
restored by shaking the milk vigorously, thus bringing it into 
intimate contact with the oxygen of the air. 

The production of an amylolytic enzym (diastase, amylase) is 
demonstrated by gas production on potatoes. This medium con- 
tains starch in large amounts. The starch is converted into 
maltose by diastase (amylase). Maltose may then be split by 
the action of an inverting enzym into dextrose, which is then 
fermented with gas production. 

Second page 

1. Note the reaction of the medium. 

2. The incubation temperature may be (a) 37 C. 
(thermostat), (b) room temperature (20 C.), 1 (c) ice 
chest. 

1 Gelatin cultures are to be incubated at room temperature. 



LABORATORY GUIDE IN BACTERIOLOGY 






FIG. 21 
Streak Cultures 



f 





BACTERIOLOGICAL TECHNIC 




FIG. 23 
Liquefaction of Gelatin 




1. Gas bubbles in glucose agar 

2. Coagulation of milk 



3 4 

FIG. 24 

3- Coagulation and peptonization of milk 

4- Complete peptonization of milk 



66 LABORATORY GUIDE IN BACTERIOLOGY 

3. Plates are to be described only once after 24 or 
48 hours, according to growth. Make notes in column 
7 for agar plate; in column 8, for gelatin plate. Make 
sketches in the margins reserved for this purpose. 

4. The growth on the media i, 2, 3, 4, 5, and 6 is 
to be described fully according to the outline in the 
spaces under 24 hours. In the spaces under 48 hours 
and 6 days note only the changes from the first descrip- 
tion. 

5. Make sketches frequently and accurately, espe- 
cially from milk and gelatin media, and any other 
noteworthy growth, in the columns reserved for this 
purpose; also a sketch of each organism from part of a 
field under the microscope. 

These directions for filling out culture charts are 
applicable in the described manner to studies of cul- 
tures furnished by the laboratory. For determining 
any species of unknown bacteria, original researches 
must be made to cover all points, without the possi- 
bility of gathering this information from textbooks or 
references. On the accuracy of observation and de- 
scription depends the success of bacteriological work 
and species determination. It is frequently neces- 
sary to employ special media, or inoculation of animals, 
or such biological reactions as the agglutination test, 
to determine what species one is dealing with. 

The student will do well to familiarize himself 
with all directions and explanations given in this and 
the previous chapters. Find a characteristic for each 
item mentioned; otherwise the descriptions will be 
incomplete; and follow the instructions for routine 
work with all possible accuracy. 



PART II 
GENERAL BACTERIOLOGY 



SECTION i 
PREPARATION OF CULTURE MEDIA 

The following culture media are to be prepared for 
this work : 

FIRST SET 

Wort agar 20 tubes 

Ten of these tubes should contain about 7 c.c. for slants, the 
other ten about 10 c.c. for plating. 

Wort gelatin ' 10 tubes 

Liquid wort 10 tubes 

Fermentation tubes 6 tubes 

Two of the fermentation tubes to contain i per cent dextrose, 
two i per cent lactose, and the remaining two i per cent saccha- 
rose. 

SECOND SET 

Meat extract agar 20 tubes 

Ten of these for slants and ten for plating. 

Pepton gelatin 15 tubes 

Broth 15 tubes 

Dextrose agar. 15 tubes 

Litmus milk 15 tubes 

Synthetic potato 10 tubes 

The beerwort media are to be prepared during the 
nrst week, the other media later. 



70 LABORATORY GUIDE IN BACTERIOLOGY 



SECTION 2 

COLLECTING AND CULTIVATING MICRO-ORGANISMS 
FROM THE AIR 

EXERCISE I 

1. Sterilize all petri dishes in the hot-air sterilizer for 
one hour at 160 C. 

2. Melt two tubes of plain agar, one of dextrose 
agar, and two of beerwort agar, in a water bath. The 
water bath (Fig. 25) is a round copper vessel with a 
number of holes in the top. These holes are large 
enough to allow a culture tube to slip in. A thermom- 
eter is passed through a rubber cork with a hole in its 
center, and inserted into one of the holes in the water 
bath. Water is then poured into the apparatus until 
the level is slightly higher than the media in the tubes 

and the thermometer 
lowered until the mer- 
cury bulb is immersed 
in the water. The cul- 
ture tubes are then 
slipped in, and the 
water heated to 100 C. 
3. Singe the cotton 
stopper of the liquefied 
agar tubes in the flame, 
remove the cotton 
stopper, pass the mouth 
and about one inch of 
FlG 2S the tube through the 

Water Bath , flame, and pour the 




GENERAL BACTERIOLOGY 71 

contents into a sterile petri dish, carefully lifting the 
cover (Fig. 26) and quickly replacing it. 

4. Repeat this operation with the other tubes, 
excepting glucose agar. 

5. Place all petri dishes containing the liquid agar 
on a level surface. 

6. When the agar has solidified, expose, by removing 
the cover, one dish of plain agar and one of wort agar 
to the air of the laboratory, and the other two outside 
on the window-sill, for 5 minutes. 

7. Replace the cover and place in incubator. 




FIG. 26 
Pouring Medium into Petri Dish 

8. Cool the water bath to 43 C., and mix the 
scrapings from under a finger nail with the liquid 
glucose agar. Incubate at 37 C. 

9. Remove the plug of a tube of broth and place a 
hair in the liquid. Incubate at 37 C. 

Liquefied agar media should be inoculated at a 
temperature no higher than 43 C. nor lower than 40 C. 
Above 43 C. the organisms are liable to be injured 
by heat; below 40 C. the agar solidifies, and an even 
distribution is impossible. If gelatin is used, the 
latter precaution is not imperative, as gelatin solidifies 
at about 25 C. 

Observe and make notes on the appearance of these 
petri dishes after 24 hours. By this time it will be 



72 LABORATORY GUIDE IN BACTERIOLOGY 

observed that a number of spots of different sizes, 
shapes, colors, etc., have formed on the surface of the 
medium. 

The low power of the microscope will reveal a dark 
part in most of these spots. This dark part is a dust 
particle, which has carried micro-organisms to the 
surface of the agar, and thus opportunity is given for 
multiplication and formation of a "colony." A colony 
originating in this manner may or may not consist of 
one species of organism. By studying the organisms 
we will find that the colonies are composed of bacteria, 
yeasts, torulae, or molds. All or any of these may be 
carried by dust particles in the air. 

EXERCISE 2 

Inoculate two or three colonies on agar slants. 

Method of inoculation 

1. Singe the cotton plug of a tube containing the 
medium. Organisms in the air are constantly alighting 
on the cotton, and bacteria may also be deposited on the 
cotton by handling it with the fingers. If these organ- 
isms are not killed by the process of singeing, they may 
drop on the medium after removal of the stopper, and 
thus ruin a pure culture. 

2. Hold the tube (or, if a transfer is made, both 
tubes side by side) between the thumb and the fore- 
finger, so that the end of the tube rests on one edge of 
the hand (Fig. 27), holding them at an angle of about 
45. If held horizontally, the condensation water, 
usually present at the lower end of the agar slant, will 
moisten the surface and destroy a characteristic growth 



GENERAL BACTERIOLOGY 73 

along the needle track. If held in a vertical position, 
contamination from the air may take place. 

3. Remove the cotton stoppers by taking hold of 
the singed portion, and hold also by the singed part 
between the other fingers. If the portion of cotton 
from the inside of the tube is touched by the fingers, or 
accidentally falls on the table or the floor, it becomes 
contaminated and must be singed before replacing. 

4. Sterilize the straight platinum needle in the 
flame, holding it like a pencil. The platinum wire 





FIG. 27 
Method of Inoculating Media 

should be heated until red hot, and the glass end passed 
slowly through the flame once or twice. 

5. After cooling the needle by plunging into the 
medium, take up a small portion of a colony or culture 
on the end of the needle by a lateral movement, and, 
when removing it from the tube, take care not to touch 
the walls of the tube. If any cotton accidentally sticks 
to the mouth of the tube, it should be burnt off with a 
hot platinum needle, and then the mouth of the tube 
passed through the flame before inserting the needle. 



74 LABORATORY GUIDE IN BACTERIOLOGY 

6. Insert the needle with the culture into the sterile 
tube and draw along the surface of the slant, without 
puncturing the jelly, and turn the needle during the 
operation. 

7. Replace the cotton plugs and sterilize the needle 
in the flame, as above. 

A stab culture is made by puncturing the medium 
centrally by a quick, steady movement of the needle. 
Care should be taken not to let the needle touch the 
bottom of the tube, the object being to make a narrow 
puncture, and by touching the glass the needle would 



----"r.- c 



a. Hollow 



FIG. 28 

Hanging Drop 
b. Drop c. Cover slip 



d. Slide 



bend and make a ragged opening in the medium on 
withdrawing. 

Inoculations of liquid media are made by rubbing 
the end of the needle against the glass below the surface 
of the liquid. The medium is then shaken. After 
inoculation liquid cultures should not be shaken again, 
as this might destroy a characteristic shape of the 
coagulum, break up the cream ring, or defeat sedi- 
mentation in broth. 



GENERAL BACTERIOLOGY 75 

Inoculations from liquid media are made with the 
looped needle. This may also be used from a solid 
medium, if a considerable amount of growth is required. 
The contents of the loop are then spread over the whole 
surface. 

EXERCISE 3 

Describe the appearance of the colonies as outlined 
in Section 9. 

EXERCISE 4 

Examine as to motility in the hanging drop. 
Preparation of a hanging drop (Fig. 28) : 

1. Clean a cover slip in alcohol. Pass several 
times through the flame so as to burn the last traces of 
grease off the surface. 

2. Place a loopful of pure water on the center of the 
cover slip. 

3. Flame the straight platinum needle, and, after 
cooling, touch one of the colonies and mix lightly with 
the drop of water without spreading it. Take only a 
minute amount of culture, so as to produce a faint 
cloudiness in the water. 

4. Smear vaselin around the depression in a hollow- 
ground slide, invert the cover slip over the depression, 
and gently press the margin on the vaselin. 

5. Examine in oil, using very little light, 

EXERCISE 5 

Molecular movement. Rub a small amount of car- 
min in a mortar with some water and make a hanging- 
drop preparation. When examining this through the 
oil-immersion lens, it will be observed that the small 



76 LABORATORY GUIDE IN BACTERIOLOGY 

particles of carmin have a lively vibrating motion. 
This is called "molecular movement," "Brownian 
movement," or "pedesis." The particles scarcely 
change their relative position. Actively motile or- 
ganisms, on the contrary, change their relative posi- 
tions. The movement of these may be slow, snake- 
like, or like a fish swimming; or they may dart rapidly 
across the field. 

Now observe and describe what changes have taken 
place in the tube of broth with the hair. Compare 
with a sterile tube, noting the turbidity, sediment, odor, 
etc. Also examine in hanging drop. 

Examine the tube of glucose agar containing the 
nail scrapings. Describe the general appearance, and 
note whether gas bubbles are present. 

EXERCISE 6 

Make stained preparations of three different 
colonies, using the three stains: i.e., gentian violet, 
methylene blue, and carbol fuchsin, also a Gram stain. 

Method of making stained preparations 

1. Clean and flame a cover slip, or, if preferred, a 
slide may be used for this purpose. Cover slips, if 
handled by the fingers, should be held by the edges. 
Use as much as possible the forceps made for that pur- 
pose. After handling, the forceps should be sterilized 
in the flame. 

2. Place one loopful of water on the cover slip. 

3. Take a small quantity of the colony or culture 
on a platinum needle and mix with water until faintly 
cloudy. Burn the remainder of the culture off the 
needle. 



GENERAL BACTERIOLOGY 77 

4. Spread over the cover slip by two or three sweeps 
of the needle. The water should spread easily and not 
run together. If the water does not spread well the 
cover slip has not been sufficiently cleaned. 

5. Dry by moving high over the flame. 

6. Pass rapidly three times back and forth through 
the flame. This process precipitates albuminous matter 
and causes the bacteria to adhere firmly to the glass. 

NOTE. The same object may be accomplished by allowing 
absolute alcohol to evaporate from the cover slip. This method 
has some advantages, since the bacteria do not shrink from the 
heat. 

7. Cover with stain for 10-15 seconds. . 

8. Wash in water. 

9. Blot with filter paper, dry in the air or high over 
the flame, and mount in Canada balsam. 

10. Label and preserve this preparation. 

Try to avoid the mistake, made by most beginners, 
of taking too much growth on the needle. For hang- 
ing-drop preparations less material should be used than 
for stained preparations. 

EXERCISE 7 
Method of making preparations according to Gram 

1. Prepare a film of the organism to be examined, 
as for the ordinary stained preparation. 

2. Cover with gentian violet for i minute. 

3. Wash in water, and remove the water by means 
of filter paper, leaving the surface moist. 

4. Cover with Gram's iodin solution for 2 minutes. 

5. Pour Gram's iodin solution off and, without 
washing, place in a staining dish, film side up, and 
cover with 96 per cent alcohol. 



78 LABORATORY GUIDE IN BACTERIOLOGY 

6. Allow to remain in alcohol, with occasional agita- 
tion, for at least 4 minutes, or until no more stain is 
taken up by the alcohol. 

7. Dry without washing, and mount. 

This stain is an important means of differentiating 
species of bacteria. 

It is a positive Gram stain if by application of this 
method either the organism loses none of the stain or 
the stain is dark blue or dark slate blue. It is a 
negative stain if either the coloration is completely 
gone or only a light bluish tinge is left. 

The preparation before mounting may be washed in 
water and counterstained with Bismarck brown. This 
method shows all foreign matter brown in contrast to 
the bacteria, and is especially adapted for staining bac- 
teria in tissues, sputum, etc. 

By mounting a Gram stain and a gentian violet 
stain of the same organism on the same slide both 
time and material are economized. 

SECTION 3 

STUDY OF MOLDS, YEASTS, AND TORULAE 
EXERCISE I. CULTURAL STUDIES 

Yeasts, torulae, and molds grow better in a medium 
of acid reaction than in a neutral or alkaline medium. 
Media prepared from hopped beerwort are generally 
used for this purpose. 

Make transfer of a stock culture of Saccharomyces 
cerevisiae or any other species of yeast which may 
have appeared on the plates prepared in the previous 
section. Also transfer two species of molds from these 
plates to slanted wort agar. 



GENERAL BACTERIOLOGY 79 

NOTE. Molds may be recognized by the filamentous, cotton- 
like form of the colonies. The hyphae extending into the air 
carry spores (conidia). By gently touching these with a sterile 
platinum needle, the spores may be transferred to an agar slant, 
and development will take place. Colonies of yeasts or torulae 
appear smooth, moist, opaque, elevated, and slightly yellowish- 
white, or sometimes reddish. These may be transferred in the 
same manner as colonies of bacteria. Molds require careful 
handling for microscopical demonstration. They are usually 
examined in water or glycerin in the unstained condition. 

EXERCISE 2 

Method of preparing molds for microscopical 
examination 

1. Transfer some of the growth to alcohol (50 per 
cent). 

2. When thoroughly moistened, transfer some of 
the growth to a drop of glycerin on a slide. 

3. Spread carefully with a platinum needle. 

4. Cover with a slip and examine. 

5. If satisfactory, the preparation may be made 
permanent by painting a ring of asphalt around the 
edge of the cover slip. 

Molds may also be stained in the following manner: 

1. Place a small amount of mold on a slide. 

2. Cover with alcohol and allow alcohol to evapo- 
rate. 

3. Wash in water. 

4. Stain with gentian violet or methylene blue. 

5. Mount in glycerin. 

EXERCISE 3 
Study of Yeasts 

i. Examine a small amount of yeast taken from an 
agar slant in water under the high power of the micro- 



8o LABORATORY GUIDE IN BACTERIOLOGY 

scope. Note the manner of reproduction by "bud- 
ding." 

2. Prepare a culture in liquid wort of Sacch. cere- 
visiae. 

3. Pour the supernatant liquid of the 24-hour-old 
culture off, and spread the sediment on a gypsum block 
with a looped needle. 

NOTE. Gypsum blocks may be prepared in the following 
manner: Gypsum (plaster of paris) is mixed with half its volume 
of water and quickly placed in a cylinder of paper. When dry, 
the paper is cut away and the block is placed in a suitable vessel 
(a stender dish or a deep, narrow petri dish, covered by an 
inverted tumbler). The block and vessel are then sterilized in 
the hot-air sterilizer for one hour at 110 to 115 C., or in the auto- 
clave for 30 minutes. 

4. Pour enough distilled water around the gypsum 
block to submerge about one-half of it. 

5. Incubate at 25 C. 

6. Set aside in a cool, dark place for 3 or 4 days. 

7. Examine a small portion of the film on the surface 
of the gypsum under the microscope in water. 

NOTE. Under favorable conditions, and in the presence of 
oxygen, yeasts will develop spores. The porosity of the gypsum 
block, which admits free communication with the water, and the 
fact that the surface of the block is exposed to the air, offer 
favorable conditions for spore formation, which takes place in 
3 or 4 days. 

EXERCISE 4. CULTURE STUDIES OF YEASTS AND MOLDS 

Make transfers from all agar cultures of yeasts to 
wort gelatin and liquid wort. Mark these cultures 
with labels or glass pencils on the side of the tube op- 
posite to the slanted surface and just below the cotton 



GENERAL BACTERIOLOGY 81 

stopper. Incubate the gelatin cultures at room tem- 
perature, all other cultures at 25 or 37 C. in the incu- 
bator. Make descriptions after 24 hours, 48 hours, 
and 6 days, as outlined in Section 9. 

EXERCISE 5 

Select two species of yeasts and inoculate three 
fermentation tubes with each species, using the looped 
needle, so as to have each species act on the three differ- 
ent sugars. Measure the gas evolved after 24 hours 
and after 48 hours by means of Frost's fermentation 
chart or gasometer (back cover). The chart is to be 
placed between the open arm and the bulb and moved 
until the extreme upper end of the closed arm is level 
with the top of the chart and parallel with the vertical 
lines on the chart. Express the results in percentages 
as read from the gasometer. 

Gas production is not a constant accompaniment 
of fermentations. Carbohydrates are fermented by 
many organisms without gas formation, the usual 
product being an acid, often lactic acid. Such fer- 
mentations produce turbidity, but no gas. Growth 
therefore must be described and can be observed by 
turbidity, forming either in the closed arm, the bulb, 
or both. Observations are to be noted in the space 
for this purpose on the first page of the description 
charts. 

Analysis of gas produced in the closed arm 

The gas consists chiefly of carbon dioxid and hydro- 
gen, as may be proved by the following method: Fill 
the bulb with a 2 per cent solution of NaOH, and close 
the mouth with the thumb, taking care not to leave any 



82 LABORATORY GUIDE IN BACTERIOLOGY 

air between the thumb and the liquid. Now tilt the 
gas back and forth slowly from the closed arm to the 
bulb and back to the closed arm five or six times, and 
finally allow the gas to collect again in the closed arm. 
The NaOH combines with the carbon dioxid, and con- 
sequently, on releasing the thumb, the volume of gas 
will become smaller in proportion to the amount of 
carbon dioxid absorbed. The percentage of gas is 
measured again with the chart, and the relation deter- 
mined of the gas left in the arm to the original amount. 
Example 

Total percentage of gas before addition of NaOH 45 
Percentage left after absorption by NaOH .... 30 

Difference ......................... ........ 15 

30 per cent represents the amount of hydrogen and 
15 per cent the amount of absorbed carbon dioxid. 
The proportion is expressed by the formula 



co 2 15 r 

The fact that the gas remaining in the closed arm is 
probably hydrogen may be proved by tilting it into the 
bulb, previously filled with water and closed by the 
thumb. Hold a burning match over the mouth and 
release the thumb. A slight explosion takes place 
from the combination of the hydrogen with the oxygen 
of the atmosphere. 

The gas produced by yeasts usually consists chiefly 
of carbon dioxid; the gas produced by intestinal 
bacteria consists chiefly of two-thirds hydrogen and 
one-third carbon dioxid; and the gas produced by the 
proteus group consists chiefly of one-third hydrogen 



GENERAL BACTERIOLOGY 83 

and two-thirds carbon dioxid. These proportions, 
obtained by the above-described method, are but 
approximations to the actual condition. Somewhat 
different results have been reported by Keyes (Jour. 
Med. Res., 1909, N.S. 16, p. 69). For his experi- 
ments synthetic media and more precise methods for 
the control of conditions were employed. 

Gas formation by bacteria does not necessarily 
depend on the presence of carbohydrates. Nitrogen 
may be produced from nitrites and nitrates, or urea, 
hydrogen sulphid, and ammonia from proteins during 
the process of putrefaction. 

EXERCISE 6. STUDY OF THE GERMINATION OF MOLD 
SPORES 

Transfer two species of molds from the air plates 
to slant wort agar and incubate at 37 C. 

After several days, when sufficient growth has taken 
place, remove spores from the surface of the hyphae by 
means of a straight needle and suspend these in a tube 
of liquid beerwort or broth. Transfer a loopful of 
this suspension to a cover slip and examine under the 
microscope, magnifying about 600 times. If only a 
few spores are discovered in a field invert this cover 
slip over the hollow of a hollow-ground slide and keep 
in place by painting a ring of vaselin around the hollow. 
If there are too many spores on the cover slip dilute 
the suspension with beerwort or broth. This hanging 
drop is incubated and observed daily under the micro- 
scope and sketches made of the appearance. In seven 
to ten days the spores should have produced the whole 
cycle of development of the mold and new spores 
should have formed. 



84 LABORATORY GUIDE IN BACTERIOLOGY 



SECTION 4 

BACTERIOLOGICAL EXAMINATION OF WATER, AIR 
AND MILK 

EXERCISE I. BACTERIOLOGICAL ANALYSIS OF WATER 
References 

Prescott and Winslow, Elements of Water Bacteriology, New 

York, 1914. 

Savage, The Bacteriological Examination of Water Supplies, 
London, 1906. 

A bacteriological examination of water is made for 
the purpose of determining 

1. Bacterial numbers. 

2. Bacterial species. 

3. Sewage pollution. 

Collection of samples. Procure wide-mouthed, 
glass-stoppered bottles, having a capacity of at least 
100 c.c. After cleaning and drying, tie lead foil or 
filter paper over the stopper, wrap the bottles indi- 
vidually in paper and sterilize in the hot-air oven for i 
hour at 160 C. ; then deposit them in a metal or wooden 
case. The samples from surface waters should be taken 
at least one foot below the surface, to avoid contamina- 
tion with organisms from the air. If possible, samples 
should be plated on the spot or in the laboratory within 
an hour at the latest. But when a greater interval of 
time must occur, the samples should be taken to the 
laboratory packed in ice, despite the probability of 
thus partially altering the bacterial flora. 

Method of examination. A number of pipettes of 
various sizes (i c.c., 2 c.c., 5 c.c., and 10 c.c.) are plugged 



GENERAL BACTERIOLOGY 85 

with cotton and sterilized in the hot-air oven. Then a 
number of Erlenmeyer flasks are filled with 100 c.c. of 
distilled water, and these are sterilized hi the autoclave 
at 120 C. for 5 minutes. During sterilization a small 
variable amount of water is lost. This has to be 
disregarded. 

Method of procedure 

1. With a sterile pipette carry over to one of these 
flasks i c.c. of the sample after shaking. The dilution 
is now i : 100. Mark with a glass pencil. 

2. With a sterile 10 c.c. pipette remove 10 c.c. from 
another dilution flask, and add to the remainder 10 c.c. 
of the first dilution. We now have a dilution of i : i ,000. 
(See appendix.) Make a number of dilutions in this 
manner, carrying the dilutions higher in proportion 
to the quality of the water to be examined. 

3. Melt a number of agar and gelatin tubes, corre- 
sponding to the number of dilutions made, and cool to 
43 C. 

4.. Transfer i c.c. of each dilution flask to a petri 
dish. 

5. Pour the contents of one agar tube on the petri 
dish and mix this with the i c.c. of water by tipping 
the dish back and forth. 

6. Incubate the agar plates at 37 C. and keep the 
gelatin plates at room temperature. 

Estimation of colonies. The colonies are then 
counted after 48 hours, by means of a colony counter 
(see back cover). Plates should be counted which 
contain no more than 200-300 colonies. If it is neces- 
sary to count plates with a large number of colonies, 
an estimate must be made by counting different sec- 



86 LABORATORY GUIDE IN BACTERIOLOGY 

tions of the plate counter and averaging the result for 
the whole plate. 

Species determination. If the different species of 
bacteria are to be studied, the colonies must be ex- 
amined by the naked eye and the low power. Then 
those which appear to be different are transferred to 
slant agar tubes, and from these to the ordinary media. 

Sewage contamination. If Bacillus coli and strep- 
tococci are present in relatively large number, sewage 
pollution is indicated. 

Method of examination for B. coli and strepto- 
cocci 

1. i c.c. of the sample, or, if necessary, of the 
diluted sample, is added to each of a series of ten 
fermentation tubes, containing sterile i per cent 
dextrose broth. 

2. Place in thermostat. 

3. Examine after 12-18 hours. 

4. Examine a loopful of the sediment in a stained 
preparation. 

Example. If i c.c. of the sample is added to each 
fermentation tube, and six show gas formation, there 
would be six colon bacilli in each 10 c.c. if undiluted 
water is employed. By this method number of B. coli 
per cubic centimeter may be estimated. 

Formula for determining the number of B. coli 
present in water: 

= = Number of B. coli in i c.c. water. 

N=Number of tubes with gas; D = Dilution; I = Total 
number of tubes inoculated. 



GENERAL BACTERIOLOGY 87 

The presence of streptococci is determined by mak- 
ing Gram stains from each of the fermentation tubes 
after two or three days. The number of streptococci 
can be determined approximately by the same method 
of calculation as for B. coli, by substituting the number 
of tubes containing streptococci for N. 

Isolation of B. coli and streptococci is accomplished 
by plating from the fermentation tubes in lactose 
litmus agar. 

EXERCISE 2. THE BACTERIOLOGICAL EXAMINATION 
OF AIR 

For precise methods see: 

"Report of the Committee on Standard Methods for the 
Examination of Air," Am. Jour. Public Hygiene, 1910, 20, p. 
346. 

Rettger, Jour. Exp. Med., 1910, 22, p. 461. 

An approximate determination of the number of 
bacteria in the air can be made by the following simple 
method: Place 50 c.c. of broth in an Erlenmeyer flask 
(Fig. 29, a). This flask is provided with a rubber 
stopper (6) with two holes, through which two glass 
tubes (c with a wide opening and d) lead. Cotton 
plugs are then inserted at c and d, and the apparatus 
is sterilized in the autoclave. A large bottle (/), con- 
taining 5 liters of water, is then provided with a rubber 
stopper, and also with two glass tubes (g and h) ; h is 
connected with a short piece of rubber hose and a 
pinchcock (i). When the Erlenmeyer flask and con- 
tents are sterilized, the tube d is connected, by means 
of the rubber hose e, with g, and the plug at c is removed. 
By opening the pinchcock i, 5 liters of air are aspirated 



88 LABORATORY GUIDE IN BACTERIOLOGY 

through the broth in flask a. The flask is then dis- 
connected, and i c.c. is plated in agar and i c.c. in 
gelatin. The former is incubated at 37 C., and the 
latter kept at room temperature. After 48 hours the 
colonies are counted, and the result is multiplied by 
50. This then represents the amount of bacteria in 
5 liters of air. 




FIG. 20 
Apparatus for Determining the Number of Bacteria in a Definite Volume of Air 

a. Erlenmeyer flask /. Five-liter flask 

b. Rubber stopper g, k. Glass tubes 
c t d. Glass tubes i. Pinchcock 



EXERCISE 3. BACTERIOLOGICAL STUDY OF MILK 

The method for determining the number of bacteria 
in milk is fundamentally the same as for water, except 
that dilutions must be carried higher, as milk generally 
contains larger numbers of bacteria. 

Sterilization and pasteurization of milk. Some of 
the germs in milk are saprophytes (which under favor- 



GENERAL BACTERIOLOGY 89 

able circumstances produce disagreeable odors or 
tastes), and such pathogens as the bacillus of tuber- 
culosis (which may be derived from the cow, or may be 
an accidental contamination), the typhoid bacillus, 
the bacillus of "summer complaint" in children (pos- 
sibly identical with the bacillus of epidemic dysentery), 
the germs of cholera, diphtheria, and scarlet fever. 
All these, except B. tuberculosis, flourish in milk at its 
ordinary temperature. 

None of the methods employed in sterilizing milk 
render it sterile in the bacteriological sense of the word, 
but by means commonly employed most of the non- 
sporing pathogenic bacteria are destroyed, along with a 
large number of saprophytes, thus rendering milk 
comparatively safe and less subject to ordinary fer- 
mentative changes. 

i. Sterilization at 100 C. for 30 minutes. Such 
milk, if chilled and kept at a low temperature, will 
remain unchanged for more than a week, but, by 
heating, certain alterations have been produced in 
taste and appearance. 

2. Pasteurizing milk. The changes occurring in 
milk, as above mentioned, begin at about 80 C. Pas- 
teurization at a low temperature is accomplished by 
raising the temperature to 60-65 C. for a period of 20 
minutes. This has been shown to be sufficient to kill 
the germs of tuberculosis, typhoid fever, cholera, 
diphtheria, and pyogenic cocci. Spores are not killed. 
As shown by Theobald Smith, tubercle bacilli, when 
suspended in distilled water, physiological salt solution, 
broth, and milk, are destroyed at 60 C. in 15-20 min- 



90 LABORATORY GUIDE IN BACTERIOLOGY 

utes; but, if milk containing tubercle bacilli has its 
surface exposed to the air when heated to 60 C., the 
pellicle which forms on its surface may contain living 
tubercle bacilli after an exposure of 60 minutes. 

Study of the effect of the above two methods of 
sterilization as compared with each other and with 
unsterilized milk: 

1. From the fresh milk provided make three agar 
plates, using i, 2, and 3 loopfuls, respectively. 

2. Fill about 10 c.c. into each of ten sterile culture 
tubes, and keep one at room temperature and one in 
the thermostat. 

3. Treat four of these tubes in the following manner: 
Place water in a saucepan sufficient to cover completely 
the milk when the tubes are immersed in it. Raise the 
temperature to 65 C., and keep it there by regulating 
the flame. The tubes of milk are then immersed in the 
water, and kept there for 30 minutes, as it requires 
about 10 minutes for the milk in the tubes to reach the 
temperature of the water. The tubes are then taken 
out and cooled quickly by standing them in cold water. 
Place one of the tubes at incubator and the other at 
room temperature. Aerate the other two by shaking 
vigorously for i| minutes. Keep one of these at room 
temperature, the other in the thermostat. 

4. Place two more milk tubes in the arnold at 
100 C. for 30 minutes. Keep one at room tempera- 
ture and one in the thermostat. 

5. The remaining two tubes autoclave at 120 C. 
for 5 minutes, and place one in the thermostat and keep 
the other at room temperature. 



GENERAL BACTERIOLOGY 91 

6, Note the conditions of these ten tubes after 2 or 
3 days. Compare the results, and tabulate them. 
Note especially coagulation, time elapsed before coagu- 
lation sets in, gas formation, condition of whey, film, 
and odor. 

Plates in lactose litmus gelatin should be made from 
each of these tubes, and the colonies studied and 
counted. Subcultures on agar slants may also be made 
and the usual media inoculated from these, if the indi- 
vidual species are to be studied. 

SECTION 5 

EXERCISES ON INFECTION AND STERILIZATION 
EXERCISE I. PHENOMENA OF INFECTION 

1. Prepare three agar plates. 

2. Touch the surface of the jelly in one plate with 
the tips of the fingers. 

3. Touch the surface of the jelly of another plate 
with the tips of the fingers, after washing the hands. 

4. Catch a fly and allow it to walk on the surface of 
the jelly of the third plate. Release the fly and replace 
the cover. 

5. Place these three plates in a locker or thermostat 
for 24 hours. Observe and describe the results. Make 
hanging-drop and stained preparations of some of the 
colonies formed. 

EXERCISE 2. PHENOMENA OF STERILIZATION 

1. Make an infusion of hay in a flask with cotton 
stopper. (See p. 41.) 

2. Set the flask aside for 24 hours in a warm place, 
and observe the results. 



92 LABORATORY GUIDE IN BACTERIOLOGY 

3. Expose a tube of unsterilized broth to steam in 
the arnold, another to steam in the autoclave, at 
120 C. for 5 minutes each 

4. Set aside in a thermostat, and observe the results. 

EXERCISE 3. PHENOMENA OF STERILIZATION (CON- 
TINUED) 

Action of Berkefeld and cotton filters. Berkefeld 
filters are made of diatomaceous earth, and are porous 
so as to allow the passage of fluids, while retaining 
suspended solids, bacteria, etc. Some bacteria, known 
as "ultramicroscopic bacteria," are so small that they 
pass through Berkefeld filters. 

1. Arrange a Berkefeld filter so as to connect with a 
suction pump, and filter a quantity of unsterilized 
broth (Fig. 3). 

2. Set aside, and observe results. 

The filter (0), after having been connected with 
the flask, is sterilized in the autoclave. The cotton 
plug c prevents the air, which is sucked back, from 
carrying germs into the flask. The flask d is an 
intercepting or reflux flask, which guards against the 
broth becoming contaminated from water being sucked 
back if the pressure suddenly diminishes. 

EXERCISE 4 

Arrange a cotton filter as shown in Fig. 30. Vessel 
a, provided with a rubber stopper (b) with two holes, is 
arranged so as to have a glass tube (c) reach to the 
bottom. This tube is provided with cotton at the top 
opening (d) and some nutritive medium (broth, e) is 
placed inside. Through the other hole a bent glass 
tube leads out, and this tube is also provided with a 



GENERAL BACTERIOLOGY 



93 



cotton filter at /. The whole apparatus is then steril- 
ized in the autoclave at 120 C. for 5 minutes, and 
connection is made through the flask (g) and the tube 
(h) with the aspirator. Now aspirate some air through 
the flask, disconnect at /, and set aside. Observe the 




FIG. 30 
Action of Cotton Filter 



a Erlenmeyer flask 

b. Rubber stopper 

c. Glass tube 

d. Cotton filter 



e. Broth 

/. Bent glass tube with cotton filter at/ 
g. Erlenmeyer flask 
h. Tube connecting with aspirator 



results. Remove the cotton filter (d), and drop it 
into a flask containing sterile broth; place in the 
thermostat for 18-24 hours; examine and note the 
conditions then present. 

Results of the exercises in this chapter are to be 
observed by noting the appearance of colonies or 



94 LABORATORY GUIDE IN BACTERIOLOGY 

turbidity, and by preparing stains with gentian violet 
from small amounts of the material in the culture 
media. 

SECTION 6 

INFLUENCE OF DISINFECTANTS, LIGHT, AND HEAT 
ON THE GROWTH OF MICRO-ORGANISMS 

For precise methods see: 

Anderson and McClintic, Hyg. Lab. Bull. 82, 1912. 
Amer. Jour, of Public Health, October, 1912. 
Rideal and Walker, ibid., June, 1913. 
North Dakota Agricultural College, Special Bulletin, July, 
August, 1913. 

EXERCISE I 

1. Prepare fifty-seven Hill's test rods. These are 
prepared in the following manner: Glass rods about 
two inches longer than ordinary culture tubes are 
marked with hydrofluoric acid or a glass cutter (dia- 
mond or file) by a circle one inch from the end. A 
wad of cotton is then wrapped around the middle of 
the rod, and inserted in a culture tube. The rod is 
then pushed down until it nearly reaches the bottom. 
That part of the rod which is free at the upper end is 
used for labeling. The whole apparatus is sterilized 
in the dry-air oven. 

2. Fill two wide-mouthed flasks, one with 100 c.c. 
of a 5 per cent solution of carbolic acid, the other with 
loo c.c. of a i per cent solution. 

3. Fill two similar flasks, one with 100 c.c. of a solu- 
tion of mercuric chlorid i : 1,000, the other with a solu- 
tion of i : 10,000. 



GENERAL BACTERIOLOGY 95 

4. Fill two similar flasks, one with 100 c.c. of a 10 
per cent solution of formalin (40 per cent formalde- 
hyd), the other with a i per cent solution. 

5. Prepare 48-hour broth cultures of Staphylococcus 
aureus, Bacillus coli, and B. typhosus from stock 
cultures. 

6. Dip nineteen of these rods into each of these 
cultures respectively, to the depth of one inch; set 
them aside in their tubes to dry over night in the ther- 
mostat after marking each tube. 

EXERCISE 2 

We have now six flasks containing different solutions 
of disinfectants. 

1. Place in each one of these flasks nine of the pre- 
pared rods, three of which have been dipped in the 
Staph. aureus culture, three in the B. coli culture, and 
three in the B. typhosus culture. 

2. Take three rods (one of each organism) out of 
eacri flask after the lapse of half a minute, wash by 
pouring sterile physiological salt solution over them 
into a dish containing mercuric chlorid solution i : 1,000, 
and place each rod in a tube of sterile broth. 

3. Repeat the proceedings of step 2 with a second 
series of rods after 2 minutes. 

4. Repeat again after 5 minutes with the remaining 
series. 

5. Place all tubes (fifty-seven) in the thermostat. 
Three of these tubes have not been dipped into any one 
of the six flasks containing antiseptics, and are incu- 
bated with the others as controls. 

6. Observe the results on each of the four successive 



96 LABORATORY GUIDE IN BACTERIOLOGY 

days, and on the last day prove the relative growth 
by making agar plates with i c.c. of each culture, 
and count the colonies after 24 hours. 

7. Tabulate the results, and state conclusions. 

EXERCISE 3. INFLUENCE OF SUNLIGHT 

Experiment I 

1. Inoculate a flask containing 100 c.c. of sterile 
water with B. coli. 

2. After thoroughly shaking, take i c.c. by means of 
a sterile pipette, and plate in agar. Place the plate in 
the thermostat. 

3. Expose the flask to sunlight for several hours. 

4. Make another plate with i c.c. of the suspension, 
and place in a thermostat. 

5. After 48 hours count both plates, and compare 
the results. 

Experiment 2^- 

1. Melt a tube of agar and cool to 43 C. 

2. Inoculate with B. coli (or any other organism). 

3. Pour into a sterile petri dish. 

4. After solidification, turn bottom side up, and 
paste a strip of black paper on the glass, covering part 
of the surface. 

5. Expose to direct sunlight for several hours, and 
note the result. 

EXERCISE 4. INFLUENCE OF MOIST HEAT 

Read the methods of determining the thermal death- 
point of bacteria in the textbook. 

i. Prepare six broth cultures each of B. coli and B. 
subtilis. 



GENERAL BACTERIOLOGY 97 

2. Place four cultures of each organism in the water 
bath and heat. 

3. Remove one of each at 40 C., one of each at 
60 C., one of each at 80 C., and keep one of each for 
10 minutes at 100. 

4. Place one tube of each organism in the autoclave, 
and heat to 120 C. for 5 minutes. 

5. Now place all twelve tubes in the thermostat, 
including one of each organism as a control. 

6. After 24 hours, make plates of each tube in agar, 
and place them in the thermostat. 

7. After 24 hours, count the colonies and compare 
the results. 



SECTION 7 

STUDY OF CHROMOGENIC BACTERIA 
EXERCISE I. CULTURAL STUDIES 

References to chromogenic bacteria, and the pro- 
duction and chemistry of pigments : 

Fischer's lectures, Fliigge, Die Mikroorganismen. 
Lehmann and Neumann. 

Members of this group are widely disseminated in 
the air, water, etc. A few representatives will be 
studied. 

i. Inoculate agar slants from laboratory cultures of 
Bacillus prodigiosus, B. pyocyaneus, B. violaceus, and 
Sarcina lutea. Inoculate three slants each of B. 
prodigiosus and B. violaceus, and one each of Sar. 
lutea and B. pyocyaneus. 



98 LABORATORY GUIDE IN BACTERIOLOGY 

2. Label each tube with the name of the culture 
inoculated, and the date of inoculation. 

3. Place one culture of each organism in the thermo- 
stat, one culture of B. prodigiosus and B. violaceus in 
the locker, and leave the others exposed to sunlight. 

4. After 24 hours compare the growths of B. 
prodigiosus and B. violaceus, under the various condi- 
tions, in respect to 

a) Relative amount of growth. 

b) Relative amount of pigment produced. 

5. Note the characteristics of the pigments: Are 
they diffused through the medium, or are they con- 
fined to the growth ? 

6. Make descriptions of agar cultures; also hanging- 
drop, stained, and Gram preparations. 

7. Transfer from 24-hour-old agar cultures of all 
organisms to all media. (See Section 8.) Potatoes 
may be inoculated with the looped needle, as the sur- 
face is too rough to allow of a smooth inoculation with 
the straight needle. 

8. After all cultures have been incubated for 24 
hours, make all descriptions as outlined on pp. 53 ff. 

CAUTION. Through oversight gelatin cultures are sometimes 
placed by students in the thermostat. This defeats the purpose 
of obtaining a stab growth, as the gelatin will melt. In order 
to avoid this mistake, it is- recommended to label one tin cup or 
tumbler " Gelatin" in large letters. This will serve as a constant 
reminder that gelatin has to be kept at room temperature. 

9. Make plate cultures of the four organisms. 
Method of making plates 

i. Melt two agar tubes for each organism in the 
water bath and cool to 43 C. 



GENERAL BACTERIOLOGY 99 

2. Transfer 3-5 loopfuls (according to the intensity 
of the growth, to be judged by the degree of cloudiness) 
of the broth culture to a sterile tube of Dunham's solu- 
tion, or sterile physiological salt solution. 

3. Shake well, avoiding air bubbles as much as 
possible. 

4. Transfer 4 or 5 loopfuls from this suspension to a 
melted agar tube. 

5. Shake this carefully by rolling the tube between 
the palms of the hand. 

6. Transfer 4 or 5 loopfuls of this agar tube (2) to 
the second agar tube (3), and mix as above. 

7. More tubes may be inoculated in the same way, 
resulting in still higher dilutions if this is desirable. In 
the meantime the inoculated tubes should be replaced 
in the water bath, so as to keep them liquid. 

8. Pour the contents of the tubes, one after the 
other, into sterile petri dishes. 

9. Tip the petri dishes so as to distribute the medium 
evenly over the bottom. 

10. Label them with the name of the organism and 
the date. 

11. Set aside on a level place to solidify. 

12. When solidified, place them in the thermostat 
bottom up, in order to avoid moistening the surface 
of the agar by the condensation water dropping from 
the cover. 

NOTE. If the surface were moistened, the colonies would 
run together and the characteristic appearance be destroyed. 
Gelatin plates, on the contrary, are placed cover up. Conden- 
sation water does not form on these plates and gelatin may be 
liquefied by the organisms. The liquefied part would then fall 
from the medium on the cover and ruin the plate. 



100 LABORATORY GUIDE IN BACTERIOLOGY 

The plates prepared in the above manner should be 
studied after 24 hours, or, if not sufficiently developed, 
after 48 hours, according to directions in Section 9. 

EXERCISE 2. STUDY OF PIGMENTS 

On the sixth day take agar slant or potato cultures 
of the four chromogenic bacteria, and proceed as 
follows: Pour 96 per cent alcohol on the cultures of 
B. prodigiosus, B. violaceus, and Sar. lutea. The 
pigment should dissolve. Filter the liquids into clean 
test tubes, and, by adding a few drops of 5 per cent 
hydrochloric acid, note the change in color. Then 
add an excess of a 2 per cent solution of sodium hydrate, 
and note whether or not the color returns and is 
changed again. Then pour chloroform on a culture of 
B. pyocyaneus. This will dissolve the bluish-green 
pigment (pyocyanin). Filter, and evaporate on the 
water bath. When almost dry, place a small amount 
on a slide, and observe the small crystals of pyocyanin 
under the microscope. Note also the aromatic odor 
given off by the pigment as the solvent evaporates. 



SECTION 8 
STUDY OF MICROCOCCI 

Make transfers from laboratory cultures of Staphylo- 
coccus aureus and Streptococcus lacticus. 

Follow the outline of routine work and make descrip- 
tions of these two organisms. 



GENERAL BACTERIOLOGY I J ; ', IOC 



SECTION 9 
STUDY OF INTESTINAL BACTERIA 

EXERCISE I 

Make transfers from laboratory cultures of B. coli, 
B. suipestifer, B. fecalis alkaligenes, and B. cloacae. 

EXERCISE 2 

In addition to the usual routine study prepare 
fermentation tubes in the following manner: Meat 
extract broth or meat infusion broth is inoculated with 
a culture of B. coli to remove the muscle sugar and 
incubated at 37 C. for 24 hours. The broth is then 
boiled, the reaction made i per cent acid, and the broth 
filtered until clear. Dissolve i per cent dextrose in 
one third, i per cent lactose in another third, and i 
per cent saccharose in the remaining third. Fill these 
solutions into fermentation tubes and sterilize in the 
arnold for three successive days. Any gas which accu- 
mulates during sterilization in the closed arm must be 
tipped out while the medium is hot. When cooled 
down, inoculate the fermentation tubes with the four 
organisms, incubate for 24 hours, and then measure the 
gas in the closed arm. Replace in the incubator and 
measure the gas again after 48 hours. Finally deter- 
mine the composition of the gas as described on p. 81. 

In addition to the exercises outlined for this course 
demonstrations should be made of stains of tubercle 
bacilli in sputum, stains of diphtheria bacilli, methods 
of anaerobic cultivation of bacteria, and various 
outfits used in municipal laboratories for diagnosis. 



PART III 
IMPORTANT PATHOGENIC BACTERIA 



SECTION i 
PREPARATION OF CULTURE MEDIA 

The following amounts of culture media will be 
required in this work: 



Name of Medium 


Amount 


Number of Tubes!") 


Broth 


300 c.c. 


25 


Plain agar 


I OOO g 




Dextrose agar 


250 g. 


20 


Gelatin 


300 g. 


25 


Litmus milk 


200 c.c. 


25 


Potato .... 




f Io 


Dunham's solution 


300 c.c. 


25 












160 



The following staining solutions should be made up 
in sufficient quantities to fill ordinary staining bottles: 
Loffler's methylene blue. 
Ziehl-Neelsen's carbol fuchsin. 
Ehrlich's gentian violet. 
Gram's iodin solution. 

The work in this course should begin with Part II, 
Section 7, p. 97. 
/ 

SECTION 2 

THE PYOGENIC GROUP 
EXERCISE I. THE PYOGENIC GROUP (SUBGROUP A) 1 

Members 

Staphylococci, streptococci, Microc. tetragenus. 

Inoculate agar slants from laboratory cultures of 
Staphylococcus aureus, Staph. albus, and Strepto- 

1 This subdividing of the pyogenic group is an arbitrary 
measure designed to facilitate study, the commoner pyogens 
being studied first. 

105 



106 LABORATORY GUIDE IN BACTERIOLOGY 

coccus pyogenes. These organisms are pathogenic, 
and care must be taken to observe the rules of technic. 
Any carelessness may be followed by grave conse- 
quences. In case of accident, such as the spilling of a 
culture or infecting of the hands, disinfection e.g., 
with a solution of mercuric chlorid (i : 1,000) is neces- 
sary. 

After 24 hours' incubation of three agar slants, 
proceed with the other media as outlined in the routine 
study (pp. 50 ff.). 

Special study. Inoculate a rabbit intravenously 
with a broth culture of Staph. aureus. The ear of the 
rabbit is shaved, and washed with mercuric chlorid 
solution, followed by alcohol. Then i or 2 c.c. of a 
24-hour-old culture in broth is drawn up into a hypo- 
dermic syringe, which has been sterilized by immersion 
in boiling water for 10 minutes. The mode of holding 
a rabbit is as follows: The left arm of an assistant 
rests against the hind-quarters of the rabbit on the 
table, while the two hands hold the fore-legs. If the 
animal struggles, force should not be applied, as this 
might cause injury. The struggles may be overcome 
by wrapping the animal in a towel or some other piece 
of cloth. The needle is then inserted into the lumen 
of the lower vein (ramus lateralis posterior of the vena 
auricularis posterior), which has been pinched between 
the fingers, or by means of a forceps, so as to arrest 
the circulation. No air should be injected with the 
culture, as this will kill the animal. The hypodermic 
syringe is then withdrawn and sterilized in boiling 
water for 15 minutes. 



IMPORTANT PATHOGENIC BACTERIA 107 

After the death of the rabbit, study the lesions pro- 
duced by the organism, and make cultures on slant 
agar, and smears from the heart's blood, spleen, and 
foci of suppuration. 

DIRECTIONS FOR AUTOPSIES (SEE SKETCH, FIG. 31) 
See Mallory and Wright, Pathological Technique. 

1 . Have the instruments sterilized in boiling water. 

2. Tie the animal by the extremities on a square 
board, with the abdomen upward. 

3. Note the presence of any external lesions, such as 
swellings, ulcerations, etc. 

4. Wash with a solution of mercuric chlorid (i : 1,000) 
followed by alcohol. 

5. Lift the skin over the pubes with the forceps, and 
with the scissors make an incision along the median line 
well above the sternal notch; then diagonal incisions 
extending along the fore- and hind-legs. 

6. Cut the skin away with a moderately sharp 
knife, avoiding opening the abdominal cavity. 

7. Open the abdomen by a median incision from the 
pubes to the sternum. 

8. Remove the anterior thoracic wall by cutting 
away the ribs from below upward on each side to the 
thoracic apex. 

The viscera are now exposed. Cultures and smears 
should be made from the heart's blood, peritoneal 
cavity, spleen, liver, and localized foci of suppuration. 

Gram stains are of special value inasmuch as staphy- 
lococci are gram positive, while the tissues are more 
or less decolorized. 



io8 LABORATORY GUIDE IN BACTERIOLOGY 



SUBMAXILLARY 
GLANDS 



5TOMAC H 
-SPLEEN 
-1 KIDNEY 




KIDNEY- IM 

ADHENAL.- 
GLAIND5 



INGUINAL 
GLANDS 



FIG. 31 
Autopsy of a Guinea-Pig(Diagrammatic) 



IMPORTANT PATHOGENIC BACTERIA 109 

EXERCISE 2. THE PYOGENIC GROUP (SUBGROUP B) 

Members 

Streptococcus pneumoniae. 
Micrococcus zymogenes. 
Micrococcus gonorrheae. 

M. gonorrheae is difficult to cultivate on labora- 
tory media. It is a parasite and requires special media 
for cultivation. For these reasons it is sufficient to 
study the characteristic morphology in smears made 
from gonorrheal pus. Methylene blue or Pappenheim's 
stain (p. 45) and Gram stains should be made. 

Inoculate agar slants from laboratory cultures of 
Str. pneumoniae and M. zymogenes. 
References (M. zymogenes} 

MacCallum and Hastings, Jour. Exper. Med., 1899, 4, p. 521. 

Harris and Longcope, Centralbl. f. Bakt., 1901, 30, Abt. 
!> P- 353 (printed in English). 

Birge, Johns Hopkins Hospital Bull., 1905, 16, p. 309. 

1. Routine study. Note the microscopic appear- 
ance of both organisms and the action of M. zymogenes 
on milk and gelatin. 

2. Special study. The staining of capsules from 
a milk culture of Str. pneumoniae. Three methods 
may be applied for this stain: 

First method (Friedlander's method) 

1. Prepare a stain by the following formula: 

Glacial acetic acid i part 

Saturated alcoholic solution of gentian 

violet 5 parts 

Distilled water 10 parts 

2. Prepare a film in the usual manner from a 24- 
hour-old culture in milk. 



no LABORATORY GUIDE IN BACTERIOLOGY 

3. Cover with the stain for 10 to 15 seconds. 

4. Wash in water. 

5. Dry and mount in balsam. 
Second method (Welch's method) 

1. Prepare a film from a 24-hour milk culture by 
smearing a loopful thinly over a coverglass. 

2. Cover with glacial acetic acid for 1 5 to 20 seconds. 

3. Wash the acetic acid off with carbol fuchsin. 

4. Wash off the stain with o . 8 per cent NaCl solu- 
tion. 

5. Dry and mount in balsam. 

Third method (Rosenow's method) (Jour. Am. 
Med. Assoc.y 1911, 56, p. 418) 

This method is applicable for staining capsules in 
tissues as well as cultures. If the material is too thick 
or viscid it must be diluted with a suitable amount of 
distilled water. Cultures from agar, blood serum, etc., 
should be mixed on a cover slip with a loopful of serum. 

a) Prepare a thin film, and dry in the air. 

b) When dry, cover with a 5 to 10 per cent solution 
of tannic acid for 10 to 20 seconds. 

c) Wash in water and dry with blotting paper. 

d) Cover with carbol gentian violet or anilin gentian 
violet for one-half to one minute. Carbol gentian 
violet is prepared by mixing one part saturated alco- 
holic solution of Griibler's gentian violet with 4 parts 
of an aqueous 5 per cent phenol solution. 

e) Wash in water. 

/) Stain with Gram's iodin solution for one-half to 
one minute. 

g) Decolorize in alcohol. 



IMPORTANT PATHOGENIC BACTERIA III 

h) Stain with a saturated alcoholic (60 per cent) 
solution of Griiber's eosin. 

*) Wash in water and blot. 

j) Clear and mount in balsam. 

Whichever method is applied, the capsule should 
appear as a lightly stained zone with a well-defined 
outline around the deeply stained cell. Capsules are 
rarely demonstrable unless the organisms are culti- 
vated in media rich in proteins, or are mixed with 
serum previous to staining. 




FIG. 32 
Mouse Holder 

Special study. Inoculation of a mouse with Str. 
pneumoniae. 

1. Fasten the mouse in the holder (Fig. 32). 

2. Shave a place on the back above the tail.' 

3. Wash with a solution of mercuric chlorid 
(1:1,000), followed by alcohol. 

4. Inject 0.2 c.c. of a milk culture of Str. pneu- 
moniae. 

5. When dead, perform an autopsy, and study the 
lesions in the usual manner. 

6. Make cultures in milk and on slant agar from 
the heart's blood or the spleen. 

7. Make a capsule stain from the heart's blood, 
spleen, or other organs. 



112 LABORATORY GUIDE IN BACTERIOLOGY 

SECTION 3 
THE GROUP OF COLON-TYPHOID BACILLI 

This chapter is devoted to the study of the group 
of intestinal organisms. This collective group may 
conveniently be subdivided into three subgroups: 

Subgroup i : the colon group. This group includes 
different varieties of Bacillus coli and B. aerogenes. 

Subgroup 2 : the B. enteritidis group. This group 
includes B. suipestifer, B. paratyphosus, B. enteritidis, 
and B. icteroides. The term "intermediate" is assigned 
to this group, because it resembles in part the colon 
group and the typhoid group. 

Subgroup 3: the typhoid-dysentery group. This 
group includes B. typhosus, varieties of B. dysenteriae, 
and B. fecalis alkaligenes. 

EXERCISE I. STUDY OF SUBGROUP I 
THE COLON GROUP 

Inoculate agar slants from laboratory cultures of 
B. coli, B. coli anaerogenes, 1 and B. aerogenes. Also 
inoculate one tube of broth with B. coli for the prepara- 
tion of sugar-free broth. 

References 

Smith, The Wilder Quarter Century Book, Ithaca, 1893, p. 187. 
Smith, Amer. Jour. Med. Sci., 1895, N.S. no, p. 283. 
Rogers, L. A., Clark, W. M., and Davis, B. J., "The Colon 
Group of Bacteria," Jour. Inf. Dis., 14, p. 411. 

1 There are many varieties of B. coli, distinguished from each 
other chiefly by their ability to produce gas from various car- 
bohydrates. Most varieties produce gas from dextrose and 
lactose, some also from saccharose, and a few from dextrose only, 
while there is one variety, known as B. coli ana'erogenes, which 
does not produce gas from any one of the three sugars. 



IMPORTANT PATHOGENIC BACTERIA 113 

1. Routine study. Observe carefully the growth on 
potato of B. aerogenes. This organism produces an 
amylolytic enzym, which manifests itself by gas produc- 
tion. Gas bubbles frequently appear in the growth 
on potato. 

2. Special study. In order to test the action of 
micro-organisms on various carbohydrates, it is neces- 
sary to eliminate the small amount of sugar in ordinary 
broth introduced into it by meat extract, which 
generally contains muscle sugar (glycogen). This is 
accomplished by adding to freshly prepared broth a 
culture of B. coli, which decomposes many carbo- 
hydrates, including muscle sugar. By this method 
a sugar-free broth is prepared, which may be used as a 
solvent for any sugar desired. 

Preparation of sugar-free broth for the fermenta- 
tion tube: 

1. Dissolve by heat: 

Extract of beef 1.5 grams 

Pepton 5 grams 

in 500 c.c. water. Broth made from chopped beef 
(500 g. to i liter) may also be used for this purpose. 
One per cent pepton should be dissolved in meat in- 
fusion (see p. 31). 

2. After cooling, inoculate with a broth culture of 
B. coli prepared 24 hours previously. 

3. Set aside in the thermostat, for 18-24 hours. 

4. Boil 5 minutes (to kill B. coli), and filter repeat- 
edly through the same paper until clear. 

5. Adjust the reaction to i per cent acid. 

6. Divide into three equal parts and dissolve i per 
cent dextrose, lactose, and saccharose, respectively, in 
each part, and filter again, if necessary. 



H4 LABORATORY GUIDE IN BACTERIOLOGY 

7. Fill fermentation tubes, taking care to label 
each one properly, and sterilize in the arnold on 3 con- 
secutive days for 20 minutes or in the autoclave for 
5 minutes. 

All gas must be carefully tilted out of the closed 
arm of the tube while the fluid is warm. When sterili- 
zation is completed, inoculate one set of the fermenta- 
tion tubes with B. coli, another set with B. coli anaero- 
genes, and a third set with B. aerogenes. Inoculate 
with the straight or looped needle. 

Directions for measuring gas formation in fermenta- 
tion tubes and for analyzing the gas are given on p. 81. 
The reaction in the closed arm is not always the same 
as in the bulb. This may be ascertained by adding a 
small amount of litmus solution by means of a suitably 
bent glass tube. 

3. Special study. Test for indol and nitrites. 

a) Test for nitrites: Add to a culture in Dunham's 
solution, or, better, in sugar-free broth, successively i 
drop of each of the following solutions: 

(1) Sulphanilic acid 0.5 gram 

Acetic acid (25 per cent) 150 c.c. 

(2) a Naphthylamine chlorid o . i gram 

Distilled water 20 c.c. 

Acetic acid (25 per cent) 150 c.c. 

A yellowish-red or rose color shows the presence of 
nitrites. 

b) Test for nitrites and indol combined. 

(1) Add to a culture in Dunham's solution, or sugar- 
free broth, i or 2 drops pure sulphuric acid. 

(2) Heat gently. Rose color shows the presence of 
nitrites and indol. If no reaction takes place, add 



IMPORTANT PATHOGENIC BACTERIA 115 

(3) A few drops of a solution of o . i g. potassium or 
sodium nitrite in 1,000 c.c. water. Rose color then 
indicates the presence of indol only. 

The appearance of indol red depends on the pres- 
ence of NO 2 . This is liberated by sulphuric acid 
from nitrites, if these are produced by the organism. 
If nitrites are not produced, a small amount of a nitrite 
solution is added, which then furnishes the necessary 
material for production of NO 2 . 

Perform these tests with all the organisms of the 
intestinal group, and make control tests in sterile Dun- 
ham's solution or sugar-free broth. 

4. Special study. Make a capsule stain of B. 
aerogenes from 24-hour-old milk cultures. (For 
method see p. 109.) 

The study of B. coli is of special importance in con- 
nection with bacteriological analysis of water (see 
Part IV). The presence of this organism in large 
numbers indicates sewage contamination, and conse- 
quently bacteria such as B. typhosus and B. dysen- 
teriae may be present. 

EXERCISE 2. STUDY OF SUBGROUP II 
THE HOG-CHOLERA, B. ENTERITIDIS, OR INTERMEDIATE GROUP 

Inoculate agar slants from laboratory cultures of B. 
suipestifer, B. enteritidis (Gartner's bacillus), and 
B. paratyphosus. 
References 

B. cholerae suis: 

Moore, The Pathology of Infectious Diseases of Animals. 
McFarland, Textbook of Bacteriology. 
B. paratyphosus: 

Buxton, Jour. Med. Res., 1902, 7, p. 201. 



116 LABORATORY GUIDE IN BACTERIOLOGY 

Wells and Scott, Jour Infect. Dis., 1904, i, p. 72. 
Gushing, Johns Hopkins Hospital Bull., 1900, p. 156. 
Durham, Jour, of Exper. Med., 1900-1901, 5, p. 353. 

1. Routine study. Observe the bluish-green colora- 
tion of the cream ring in litmus milk, and make a 
test for indol in Dunham's solution or sugar-free broth. 

2. Special study. Inoculate plain sterile milk with 
B. suipestifer. After 8-10 days it will be observed 
that the milk is becoming transparent, due to a solvent 
action of the alkali produced by the organism upon the 
protein content. 

3. Special study. Inoculate fermentation tubes as 
with B. coli. Measure and analyze the gas. Compare 
the results with those obtained in the study of the colon 
group. 

4. Special study. Inoculation of a rabbit sub- 
cutaneously with B. suipestifer. Subcutaneous 
inoculations of rabbits are made in the following 
manner: An assistant, in a sitting position, places the 
rabbit back down in his lap. The head projects be- 
yond the knees of the assistant. The ears and hind- 
legs are grasped, and the animal is thus held in position. 
The hair is then cut off on a portion of the abdomen, 
and the place is treated with mercuric chlorid and 
alcohol. The skin is then pulled up, the syringe in- 
serted, and the material injected. 

After the rabbit has died, study the lesions pro- 
duced by the organism, and make smears from the site 
of the inoculation, the heart's blood, and other organs. 
Note the polar staining, i.e., stained portions at the 
two ends of the cell and an unstained area between. 
Make cultures on agar from the heart's blood and other 
internal organs. 



IMPORTANT PATHOGENIC BACTERIA 117 

EXERCISE 3. STUDY OF SUBGROUP III 
THE TYPHOID-DYSENTERY GROUP 

Use great care in handling members of this group. 

Inoculate agar slants from laboratory cultures of 
B. typhosus, B. dysenteriae (Shiga), and B. fecalis 
alkaligenes. 

1. Routine study. Study the reaction on milk, 
and test for indol. Preserve glucose agar cultures for 
two weeks for the observation of involution forms. 

2. Special study. Inoculate fermentation tubes in 
the same manner as in the two preceding groups. Ob- 
serve the absence of gas formation but note growth or 
absence of growth in both arms. Compare the results 
with those of the colon and intermediate groups. 

3. Special study. The staining of flagella. To 
demonstrate the presence of flagella on B. typhosus, 
the following methpd will give good results (Loffler's 
method) : 

a) The mordant: 

Tannic acid (20 per cent aqueous solution) . 10 parts 
Ferrous sulphate (saturated aqueous solu- 
tion) 5 parts 

Fuchsin (saturated alcoholic solution) .... i part 
Add one part i per cent NaOH solution for each 
100 parts of stain. 

b) "Prepare several cover slips by flaming them, and 
place them side by side on a piece of filter paper. 
(This paper must be burned after using.) 

c) Place 4 or 5 loopfuls of water on a clean slide. 

d) Make a light suspension in this water of B. 
typhosus from a 24-hour-old agar culture, taking care to 
stir the suspension as little as possible. 

e) Place a loopful of water on each of the cover slips. 
/) Carry over a loopful of the suspension on the 



Il8 LABORATORY GUIDE IN BACTERIOLOGY 

slide to one of the cover slips and from this to each of 
the other cover slips. 

g) Allow to dry in the air. 

h) Cover with the mordant. 

i) Heat over a small flame for i minutes while 
steam rises, or better, heat on a water bath for 5 minutes. 
Replace the evaporated mordant to prevent its drying 
on the cover slip. 

j) Wash in water. 

k) Drain the water off with blotting paper. 

/) Cover with anilin gentian violet or carbol fuchsin. 

m) Heat as before over a small flame for ij minutes, 
or better, on a water bath for 5 minutes. 

n) Wash in water. 

0) Dry and mount in balsam. 

4. Special study. Agglutination. Dried-blood 
method of Johnston: A drop of blood of a typhoid 
fever patient is obtained by pricking the lobe of the ear, 
previously cleaned and washed with alcohol. The 
blood is taken up by a piece of sterile non-absorbent 
paper or on a sterile aluminum slide. This is sent to a 
laboratory, where the blood is dissolved in physiologi- 
cal salt solution in such a manner as to obtain an 
approximate dilution of 1:25. This solution is then 
tested with a suspension of typhoid bacilli, a young 
culture of which is constantly kept on hand for this 
purpose. A loopfai of the diluted blood is mixed with 
a loopful of the suspension on a cover glass, this making 
a dilution of i : 50. The cover glass is then inverted over 
a hollow slide like a hanging drop and observation made 
after two hours' incubation at 37 C. For laboratory 
tests the serum of an animal (either a rabbit or a guinea- 
pig) which has been injected with cultures of B. typho- 



IMPORTANT PATHOGENIC BACTERIA lip 

sus, previously heated for i hour at 60 C., is used. 
This process kills the organisms, but the toxins remain 
active. The first injection is followed by another one 
with dead cultures after four to five days, and after the 
same intermission a culture of virulent bacilli is in- 
jected. By this time the agglutinative power of the 
blood is well developed. The animal is then bled in 
the following manner: One of the ears is shaved, and the 
skin is washed with alcohol. A small vein near the 
border is opened, and the blood is collected in a sterile 
glass vessel. If the animal is hung head down enough 
blood can be collected in a short time. The blood is 
placed in the ice chest, and the serum is collected after 
separation. 

The method of procedure with serum obtained in 
the above-described manner is as follows: 

a) Small quantities of the serum are diluted with 
sterile salt solution (0.85 per cent) so as to represent 
dilutions of i : 5, i : 25, and i : 50. 

b) A suspension of a 24-hour-old agar culture of B. 
typhosus in salt solution is prepared. This suspension 
should be uniform and not heavy. It is desirable to 
filter the suspension through absorbent cotton or sterile 
filter paper to remove clumps of bacteria. 

c) Three hanging-drop preparations are made by 
mixing a loopful of this suspension with a loopful of the 
three serum dilutions, respectively. The final dilutions 
then are: i : 10, i : 50, and i : 100. 

d) Examine with the high power (dry lens), and 
observe the clumping of the bacilli, preceded by the 
loss of motility. 

e) Tabulate the results as to time and completeness 
of reaction. 



120 LABORATORY GUIDE IN BACTERIOLOGY 

/) Make a control hanging drop without serum to 
test the motility and the absence of clumps. 

g) When the clumping is completed allow the drop 
to dry in the air without stirring or spreading, fix in the 
flame, or better with absolute alcohol, and stain with 
gentian violet. A permanent preparation can be 
obtained in this manner, showing the agglutinated 
bacteria. 

Blood may also be obtained by puncturing the lobe 
of the ear and collecting the blood in a capillary glass 
tube with a small bulb. Hold the bulb down, fill 
three-fourths with blood, and seal the ends in the 
flame. In 45 minutes the serum will have separated, 
and may be tested. 

The above-described method of an agglutination 
test is known as the microscopic test. Another 
method, in which larger amounts of serum and suspen- 
sion are required, is known as the macroscopic method. 
Small test tubes are used, and- definite amounts of 
bacterial suspensions are introduced, by means of 
graduated pipettes. The serum is then added in vary- 
ing amounts so as to effect the desired dilutions (see 
table in appendix, p. 197). The tubes are then incu- 
bated at 37 C., usually for 2 hours. . After this they 
are placed in an ice chest for sedimentation. If com- 
plete agglutination has taken place, the bacteria will 
have collected in clumps at the bottom, forming a 
sediment. The supernatant fluid is clear. By varying 
amounts of sediment and varying degrees of turbidity 
of the supernatant fluid, the degree of agglutination 
may be estimated. A control tube of a bacterial 
suspension without addition of serum serves as a guide. 
Controls with normal serum should also be made. 



IMPORTANT PATHOGENIC BACTERIA 121 

5. Special study. Make cover-slip preparations of 
B. dysenteriae from glucose agar cultures 10-12 days 
old. Involution forms are then plentiful and can be 
studied. 



SECTION 4 
THE PROTEUS GROUP 

Inoculate agar slants from laboratory cultures of 
Bacillus proteus, Proteus zenkeri, and Bacillus cloacae. 

1. Routine study. Observe the action on milk and 
gelatin. 

2. Special study. Make plates in gelatin and agar, 
and observe the colonies after 24, 48, and 72 hours. 
Note the appearance of the colonies "of B. proteus 
and of Prot. zenkeri on both media. 

3. Special study. Inoculate fermentation tubes and 
determine the percentage of gas formed and the gas 
formula. Compare the results with those of the in- 
testinal bacteria. 

NOTE. In order to obtain a clear picture of the differential 
characteristics of the three groups of colon-typhoid bacilli and the 
proteus group, it is recommended to tabulate the results in 
columns, as outlined below. Express positive results by the 
sign +; negative, by . Complete agglutination is expressed 
by + +; slight, by +. 



122 LABORATORY GUIDE IN BACTERIOLOGY 



HXIM. NOiivNixmooy 
















FERMENTATION OF 
CARBOHYDRATES 


Growth in 
Closed Arm 
No Gas 


qoo^S 












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Free Gas in 


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NOixoaaoaj IOONJ 














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11 ' 


B. coli 
B. coli anaerogenes 
B. aerogenes 


1 

PQ 


B. enteritidis 


1 
1 

3 

a 

pq 


B. typhosus 




1 

pq 


1 

pq 


B. proteus 
Proteus zenkeri 


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THE 
TYPHOID- 
DYSENTERY 
GROUP 


IV 
THE PROTEUS 
GROUP 



p 



IMPORTANT PATHOGENIC BACTERIA 123 

SECTION 5 
THE CAPSULATED GROUP 



Members 

Bacillus capsulatus. 
B. rhinoscleromatis. 



Inoculate agar slants from laboratory cultures of 
B. capsulatus (Friedlander's pneumo-bacillus) and B. 
rhinoscleromatis. 

1. Routine study. Observe the viscous condition 
of cultures on solid media, the consistency of liquid 
media, and the gas formation on potato. 

2. Special study. Staining of capsules from 24- 
hour-old milk cultures (see p. 109). 

3. Special study. Intraperitoneal inoculation of a 
rabbit with B. capsulatus. 

Method of intraperitoneal inoculation. The rabbit 
is held in the same manner as described on p. 106. 
The hair is clipped close over the left lower abdominal 
quadrant. Then (after washing with mercuric chlorid 
i : 1,000 and alcohol) pass the needle at first beneath the 
skin, then, holding it at about a right angle to the 
abdominal surface, push it through the abdominal wall, 
which is usually made tense by the resistance of the 
animal. Successful passage of the abdominal wall can 
be felt by the sudden loss of resistance to the needle's 
pressure. Then make the injection, and withdraw 
the syringe. If the contents of the needle have been 
emptied into the peritoneal cavity, no swelling takes 



124 LABORATORY GUIDE IN BACTERIOLOGY 

place, as is noticed in subcutaneous inoculations. 
Care is necessary not to puncture the intestines. 

When the animal has died, perform an autopsy and 
study the lesions. Make cultures from the heart and 
internal organs in the usual manner, and make capsule 
stains from the heart's blood. 

SECTION 6 

THE DIPHTHERIA GROUP 

Members 

Bacillus diphtheriae. 
B. hofmannii. 
B. xerosis. 

Use great caution in handling B. diphtheriae. 
Inoculate' agar slants from laboratory cultures of 
B. diphtheriae and B. hofmannii. 

i. Routine study. Stain B. diphtheriae with Lof- 
fler's methylene blue instead of gentian violet. The 
staining may be facilitated by the application of mild 
heat. Observe the darkly stained granules and make 
sketches of some of the bacilli. 
2. Special study. 

Neisser's Granule Stain (Gin's Modification) 
i. 5 per cent alcoholic solution. Methy- 
lene blue (Grubler) 20 c.c. 

5 per cent glacial acetic acid 1,000 c.c. 

a. 10 per cent alcoholic solution. Crys- 
tal violet (Hochst) 10 c.c. 

Distilled water 300 c.c. 

Two parts of i with one part of 2 ; call solution a. 

b. Acid lactic i c.c. 

Lugol's solution 99 c.c. 

c. Chrysoidin (i gram in hot water) .... 300 c.c. 



IMPORTANT PATHOGENIC BACTERIA 125 

Stain film for 2 seconds with solution a and wash. 
Apply solution b for 3 to 5 seconds and wash well. 
Apply solution c for 3 to 5 seconds, wash, dry, mount. 

3. Special study. Test for acid formation in a 
culture, in neutral glucose broth, one week old, by addi- 
tion of a few drops of litmus solution, or by titration 
with N-^ NaOH solution. 

4. Special study. Cultivation of B. diphtheriae 
on eggs (method of Wyatt Johnston). 

NOTE. This method is recommended as an emergency cul- 
ture test, the egg taking the place of Loffler's blood serum. 

a) Sterilize over the flame an empty meat extract 
pot, or any other vessel of suitable size. 

b) Wash a hard-boiled egg in mercuric chlorid 
solution, followed by alcohol, and break the shell at the 
blunt end with sterile forceps, without rupturing the 
membrane lining the shell. 

c) Flame the exposed part, and free the coagulated 
albumen from the membrane. 

d) Inoculate by rubbing some culture or throat 
swab on the exposed egg-albumen. 

e) Invert and set in the sterilized pot. 
/) Incubate at 37 C. 

g) Observe the appearance and make a stained 
preparation after 24 hours. 

5. Special study 
Experiment i 

a) Clip the hair over a small area on the surface of 
the abdomen of a guinea-pig or rabbit. 

b) Cut a small opening in the skin, and separate the 
skin from the muscles below by pushing in sterile 



126 LABORATORY GUIDE IN BACTERIOLOGY 

scissors. Expand these slightly and after closing 
remove. This forms a small pocket. 

c) Carry i loopful of a 24-hour-old agar culture into 
this pocket. 

Experiment 2 

a) Heat a 24-hour-old broth culture in the water 
bath for 30 minutes at 60 C. 

b) Inject o. 25 c.c. of this heated culture subcutane- 
ously into another guinea-pig (or rabbit). 

Observe and compare in both animals the results 
by taking note of the ante-mortem phenomena and the 
lesions post-mortem. 

Experiment 3 

Prepare two guinea-pigs and inject subcutaneously 
into one a lethal dose of diphtheria toxin and into the 
other the same amount of toxin neutralized with a 
sufficient amount of antitoxin. Compare the results 
in the two guinea-pigs. 

6. Demonstration of methods employed in munici- 
pal laboratories for the diagnosis of diphtheria. 

7. Study of B. xerosis. Obtain mucus from the 
inner angle of the eyelids by stroking with a platinum 
loop. Make two film preparations, stain one by Gram's 
method and the other with methylene blue. Make a 
culture on slant agar and if impure, plate out. Trans- 
fer cultures to all media from colonies on the plate and 
study these in the usual manner. 



IMPORTANT PATHOGENIC BACTERIA 127 

SECTION 7 
THE HEMORRHAGIC SEPTICEMIA GROUP 

Members 

Bacillus pestis (bacillus of bubonic plague). 
B. cuniculicida (bacillus of fowl cholera, bacillus of rabbit 
septicemia, Bacillus der Rinderseuche, Bacillus der 
Schweineseuche, etc.). 
Reference 

Moore, The Pathology of Infectious Diseases of Animals. 

Inoculate agar slants from a laboratory culture of 
B. cuniculicida or another organism of this group. On 
account of the enormous infectiousness of B. pestis it is 
not desirable to study this organism in classwork. 

1. Routine study. Stain with Loffler's methylene 
blue and anilin gentian violet. Observe ' 'polar staining. ' ' 

2. Special study. Inoculation of a rabbit sub- 
cutaneously or by scarification. When dead, study in 
the usual manner, and observe the hemorrhages pro- 
duced in the serous membranes. Make cultures from 
the heart's blood, where large numbers of bacilli will 
be found. Also make a stained preparation from the 
heart's blood, and note the typical polar staining. 

SECTION 8 

THE ANTHRAX GROUP 

Members 

Bacillus anthracis. 
B. subtilis. 

Great caution is necessary in handling B. anthracis. 
Inoculate agar slants from laboratory cultures of 
B. anthracis and B. subtilis. 



128 LABORATORY GUIDE IN BACTERIOLOGY 

1. Routine study. Observe the colonies formed on 
agar plates. Also prepare gelatin plates and study the 
colonies. 

2. Special study. Make an "impression prepara 
tion" (Klatschpraparat) from a surface colony on a 
gelatin plate. 

Method 

a) Clean and flame a cover slip. 

b) Place on a colony of suitable size, and gently 
press down, taking care not to press so hard as to 
disturb the characteristic shape of the colony. 

c) Lift the cover slip with the forceps. 

d) Dry, fix, and stain with methylene blue or by 
Gram's method. 

e) Examine under low and high power (dry lens). 

3. Special study. Staining of spores. 
Moller's method: 

a) Prepare several (five or six) films from agar 
or potato cultures of B. anthracis (or B. subtilis). 

b) Place in chloroform for 2 minutes. 

c) After drying in the air, cover with a 5 per cent 
solution of chromic acid for 2 minutes. 

d) Wash in water. 

e) Cover with carbol fuchsin and heat for 5 minutes 
on a water bath, or over a small flame. 

/) Decolorize with i per cent sulphuric acid for 25- 
30 seconds. 

g) Wash in water. 

h) Counterstain with methylene blue for 10-15 sec- 
onds without heat. 

i) Wash, dry, and mount in balsam. 

NOTE. The body of the cell should appear blue; the spore, 
red. 



IMPORTANT PATHOGENIC BACTERIA 129 

4. Special study. Demonstration of filament forma- 
tion. 

a) Spread a loopful of a broth culture of B. anthracis 
or B. subtilis on a cover glass. 

b) Dry and fix in the flame. 

c) Cover with strong acetic acid (80 per cent) for 
5-10 seconds. 

d) Wash in water. 

e) Stain with gentian violet. 

/) Wash in water, dry, and mount in balsam. 

5. Special study. Inoculate a guinea-pig subcu- 
taneously with o . 2 c.c. of a 24-hour-old broth culture of 
B. anthracis, or insert a loopful of a 24-hour-old agar 
culture in a "pocket" under the skin. When the 
animal is dead, perform an autopsy, and observe the 
hemorrhagic and gelatinous edema under the skin; also 
the enlarged spleen and the hemorrhagic adrenals. 
Make a stained preparation from the heart's blood, and 
observe the lack of spores, and also the presence of 
capsules and degenerate forms, which do not stain 
well. Make a culture on agar from the heart's blood 
or from the spleen and study the culture in the usual 
manner. 

SECTION 9 

THE SPIRILLUM GROUP 

Members 

Spirillum of Finkler and Prior. 

Sp. metchnikovii. 

Sp. tyrogenum. 

And a number of spirilla indigenous to water. 

Inoculate agar slants from laboratory cultures of 
Sp. of Finkler and Prior, and Sp. metchnikovii. The 
spirillum of Asiatic cholera is studied in Section nB. 



130 LABORATORY GUIDE IN BACTERIOLOGY 

1. Routine study. In addition to the usual media, 
inoculate an extra tube of Dunham's pepton solution 
from each organism. Observe from day to day the 
action of these two organisms on gelatin, and compare 
the results by tabulation. Observe the formation of 
coccoid involution forms on agar after 3 days. Also 
make plates in gelatin, observe the colonies from day 
to day, and compare. 

2. Special study. Test for the nitroso-indol or 
cholera-red reaction. (See test for indol, p. 114.) 
Make two tests, using one of the cultures in Dunham's 
solution after 24 hours, the other after 6 days. Com- 
pare the results of these two tests. 

3. Special study. Stain for flagella by Loffler's 
method (see p. 117). 

4. Special study. Schottelius' enriching method, 
designed to demonstrate the presence of spirilla in 
water. 

a) Prepare a solution of 2 grams Witte's pepton and 
o . 5 gram sodium chlorid in 100 c.c. of water. 

b) Distribute in three small Erlenmeyer flasks, and 
sterilize in the autoclave. 

c) Inoculate one of these flasks with Sp. metch- 
nikovii and B. suipestifer or any other motile bacillus. 

d) Incubate at 37 C. for 18-24 hours. 

e) After that time, take one loopful from the surface, 
inoculate the second flask, and incubate as before. 
Also make a stained preparation from the surface of 
the solution. 

/) After 18-24 hours, make a stained preparation 
from the surface of the second flask, and examine for 
spirilla. 



IMPORTANT PATHOGENIC BACTERIA 131 

g) Transfer a loopful from the surface of the second 
flask to the third one, and incubate as before. 

h) After 18-24 hours, again stain and examine for 
spirilla. By this time a film has formed which contains 
the spirilla practically in pure culture. 

5. Special study. Inoculate a pigeon intramuscu 
larly with 0.5 c.c. of a broth culture of Sp. metch- 
nikovii. The breast of the pigeon is laid bare, washed 
with mercuric chlorid and alcohol, and the syringe 
plunged into the muscle fibers and discharged. After 
death, note the peculiar appearance, resembling that 
of boiled beef. Make stained preparations from the 
blood and muscle juice, and examine for spirilla. 



SECTION 10 
THE GROUP OF ACID-PROOF BACILLI 

Members 

Bacillus tuberculosis. 

B. leprae. 

B. smegmae. 

Moller's grass bacillus, and a number of bacilli found on 
grass, dung, in butter, milk, etc. 

Cultural studies of B. tuberculosis consume a great 
deal of time and are, therefore, impracticable in an 
elementary course. 

For comparison, the culture characteristics of 
Moller's grass bacillus are instructive. 

Inoculate an agar slant from a laboratory culture of 
Moller's grass bacillus. 

i. Routine study. 



132 LABORATORY GUIDE IN BACTERIOLOGY 

2. Special study. Method of staining acid-proof 
bacilli. 

a) Pick out purulent matter from the sputum of a 
tuberculous patient and spread carefully on a cover 
glass. 

b) Dry and fix as usual. 

c) Heat with carbol fuchsin over a small flame for 
one minute or on a water bath for two minutes. 

d) Decolorize with acid alcohol (2 per cent HC1 in 
80 per cent alcohol) until the film has lost almost all 
its color. 

e) Wash in water and counterstain with methylene 
blue for 10 seconds (cold). 

/) Wash again and mount in balsam. 

NOTE. Make a second preparation, substituting anilin gen- 
tian violet for carbol fuchsin, and Bismarck brown for methylene 
blue. 

3. Special study. Stain Holler's grass bacillus 
from an agar culture by the same method, omitting the 
counterstain. 

4. Special study. Make a smear from a culture 
of Moller's grass bacillus in milk and stain this smear 
for acid-proof bacilli. 

SECTION nA 

MISCELLANEOUS ORGANISMS: BACILLUS MALLEI, 

BACILLUS MELITENSES, BLASTOMYCES 

DERMATITIDIS 

B. mallei has been the cause of more accidents 
among bacteriologists than any other organism, and 
it is therefore not prudent to study this organism in 
the laboratory unless there is thorough supervision. 



IMPORTANT PATHOGENIC BACTERIA 133 

The virulence of B. mallei seems not to diminish in 
laboratory cultures, and careless handling may affect all 
those who are engaged in work in the same place. 
Those who desire to study this organism will find 
directions in the following section. 

Inoculate agar slants from laboratory cultures of 
B. melitensis and Blastomyces dermatitidis. 

Carry these cultures through the usual routine. 

SECTION nB 

In this section the following organisms have been 
included: Spirillum cholerae, Bacillus mallei. Bacillus 
influenzae, Micrococcus meningitidis (meningococcus). 

A separate section has been devoted to these organ- 
isms so as to enable the instructor to omit them if he 
deems it advisable. The Sp. cholerae and B. mallei 
are dangerous organisms to be manipulated by ele- 
mentary students, and unless there is sufficient super- 
vision accidents of grave consequences are liable to 
happen. The Sp. cholerae may be studied in the 
usual routine manner and the test for the so-called 
cholera-red reaction (indol reaction) and Schottelius 
enriching method (see p. 130) added. B. mallei should 
also be studied in the usual manner and a demonstra- 
tion of its infectiousness made on guinea-pigs. 

B. influenzae requires special media for study, blood 
agar being the most suitable. The meningococcus 
grows to some extent on ordinary media. Stains 
with methylene blue and according to Gram's method 
are instructive, showing the resemblance of this organ- 
ism to the gonococcus. 



134 LABORATORY GUIDE IN BACTERIOLOGY 



SECTION 12 
PATHOGENIC TRICHOMYCETES 

Members 

Actinomyces bovis (hominis) and Actin. asteroides. 

Leptothrix. 

Cladothrix. 

Nocardia. 

1. Inoculate broth and potato from laboratory cul- 
tures, and make descriptions and stained preparations 
as usual. Other media need not be inoculated. 

2. Special study. Suspend a small amount of the 
potato culture in physiological salt solution, and 
examine under the low power. 

3. Special study. Examine a sample of actinomy- 
cotic tissue (bovine) in the fresh state, for so-called 
"sulphur granules." Crush some of the material in 
salt solution under cover slips and search for "clubs," 
using the low and high power dry lenses, and stain by 
Gram's method; counterstain with eosin or Bismarck 
brown. 

SECTION 13 
THE GROUP OF ANAEROBIC BACILLI 

Members 

Bacillus tetani. 
B. edematis. 
B. welchii. 
B. chauvei. 
B. botulinus. 
And others. 

Anaerobic cultivation involves the growth in an 
atmosphere devoid of oxygen. This is accomplished 
by removing the oxygen from air by chemicals, or 



IMPORTANT PATHOGENIC BACTERIA 135 

consuming the oxygen by burning paper, or by sub- 
stituting hydrogen gas for air. The following methods 
are most commonly in use. 

1. Park's method. 

a) Boil three tubes of dextrose agar vigorously for 5 
minutes, to drive out the dissolved oxygen. 

b) Cool to 43 C. and inoculate from laboratory 
cultures of B. tetani, B. edematis, and B. welchii. 

c) Solidify rapidly by immersion in cold water, 

d) Cover the medium with a thin layer of liquid 
paraffin or sterilized mineral oil. 

e) Incubate at 37 C. 

NOTE. The layer of paraffin or oil excludes atmospheric 
oxygen which is inhibitory to the growth of anaerobes. The 
oxygen necessary for their multiplication is derived from carbo- 
hydrates in the medium. 

2. Wright's modification of Buchner's method. 

a) Liquefy, as before, six dextrose agar tubes, 
plugged with absorbent cotton. Cool three to 43 C. 
and inoculate while fluid. Let the other three become 
solid, and make stab cultures. 

b) Sterilize the cotton stoppers in a flame, and with 
the forceps, sterilized in a flame, push the stoppers into 
the test tubes for the distance of about i inch (2-3 cm.). 

c) Pour into the tubes (upon the cotton stoppers') 
2 c.c. of a saturated solution of pyrogallic acid in water, 
followed by 2 c.c. of a 2 per cent solution of NaOH. 

d) Cork the tubes immediately with rubber stop- 
pers, and keep upside down. 

e) Incubate at the required temperature. Pyro- 
gallic acid in alkaline solution absorbs oxygen, leaving 
the cultures in an atmosphere of nitrogen. 



136 LABORATORY GUIDE IN BACTERIOLOGY 

3. Cultivation by Buchner's method, using fruit 
jars. 

a) In a Mason fruit jar of ordinary type deposit 
10 g. of pyrogallic acid. 

b) Smear vaselin around the mouth of the jar. 

c) Pour into the jar 100 c.c. of a i per cent solution 
of NaOH. 

d) Deposit in the jar culture tubes previously in- 
oculated. 

e) Fasten the cover of the jar, and incubate at 37 C. 
for 48-72 hours. 

4. Cultivation in hydrogen gas. 

a) Inoculate all media from laboratory cultures. 

b) Fit up apparatus as shown in Fig. 33. 

c) Place culture tubes in a Novy jar (Fig. 33, a). 

d) Open the faucet (b) of the gas generator (c), con- 
taining zinc and hydrochloric acid. The hydrogen gas 
generated is purified by passing through two jars, one 
of which contains concentrated sulphuric acid (d), the 
other a 10 per cent solution of sodium hydrate (e). 
Gradually the Novy jar is filled with hydrogen gas, 
which can be ascertained by holding a culture tube 
over the opening (/) and then over a burning match 
or gas flame. As long as a detonation takes place the 
hydrogen is mixed with atmospheric oxygen. When 
the hydrogen in the Novy jar is pure, close it off by 
turning the stoppers (g) and (b), and place the jar in 
the incubator. The whole process occupies 10 or 15 
minutes. 

5. A simple and effective method of anaerobic culti- 
vation is as follows: Culture tubes, after inoculation, 
are placed in a desiccation jar or any other jar with 



IMPORTANT PATHOGENIC BACTERIA 



137 



tight-fitting cover. Vaselin is smeared on the top of 
the jar to insure a tight fit of the cover. A small piece 
of absorbent paper wetted with a few drops of alcohol 
is placed inside of the jar, the paper is lighted, and the 
cover replaced. The burning paper absorbs the oxygen, 
leaving the cultures in an atmosphere of nitrogen. 

When growth has been obtained by any of the above 
methods, transfers should be made to milk, and gentian 




FIG. 33 
Anaerobic Cultivation in Hydrogen Gas 

a. Novy jar . Sodium hydrate solution 

b. Glass cock /. Opening of Novy jar 

c. Gas generator g. Stopper 

d. Sulphuric acid 

violet and Gram stains prepared. Motility should be 
determined by preparing a hanging drop. Anaerobic 
bacteria in a hanging drop will concentrate toward 
the center of the drop away from the oxygen of the air. 
6. Special study. Inoculation of a rabbit with B. 
welchii. 

a) Shave the ear of the rabbit. 

b) Wash with mercuric chlorid solution and alcohol. 



138 LABORATORY GUIDE IN BACTERIOLOGY 

c) Inoculate intravenously with 0.5 c.c. of a 24- 
hour-old milk culture of B. welchii. 

d) After the culture has been distributed in the 
circulation, which takes at the most 3 minutes, kill the 
rabbit by a blow on the back of the neck. 

e) Put the rabbit in a warm place on top of the 
thermostat for 18 hours, or from 6 to 8 hours inside of 
the thermostat. 

/) After this time has elapsed, perform an autopsy. 
Note the crackling, on pressure, over the axillary or 
inguinal regions. The rabbit is swollen to a great 
extent. Skin the animal, without opening the abdomi- 
nal cavity; then quickly puncture the abdominal wall 
and bring a flame to the opening. Note that the 
escaping gas will burn with a blue flame. Also note 
the disorganized condition of the liver, spleen, and 
kidney. 

g) Make capsule stains from the heart's blood or 
organs by Welch's method (modified). The modifica- 
tion of Welch's method is as follows: Proceed in the 
manner indicated on p. no, and, after washing the 
acetic acid off with the stain (carbol fuchsin or gentian 
violet), dry with filter paper and heat the specimen for 
5 to 10 seconds before washing. Then proceed as be- 
fore. 

7. Special study. Staining of spores of B. tetani 
and B. edematis from 3-day-old glucose agar cultures 
(see p. 128). 

8. Special study. Inoculation of a white mouse 
or guinea-pig with B. tetani or its toxin (o.oi c.c.) in 
the hind-leg or over the root of the tail (if a mouse). 
Note daily the condition of the animal, and when dead 



IMPORTANT PATHOGENIC BACTERIA 139 

make cultures and cover-slip preparations from the 
site of the inoculation. 

9. Special study. Inoculate a white rat with garden 
earth subcutaneously or in a pocket above the root of 
the tail. Note the condition of the animal daily. 
When dead, make cultures and cover-slip preparations 
from the site of the inoculation. 

SECTION 14 

ISOLATION OF UNKNOWN BACTERIA FROM A 
MIXTURE 

1. Make hanging-drop, stained, and Gram prepa- 
rations from the mixture. Note observations and re- 
sults. 

2. Melt five or six agar tubes, and cool to 43 C. 

3. Transfer 5 or 6 loopfuls of the mixture to a tube 
of liquid agar, from this to a second, and so on until all 
the melted tubes are inoculated. 

4. Pour into sterile petri dishes, and mark them 
with successive numbers and the date. Place in the 
thermostat at 37 C. 

5. After 24 hours examine the colonies under the 
low power, describe them in the usual manner, and 
transfer to agar slants all those which appear different. 

6. Transfer to dextrose agar and litmus milk and 
incubate at 37 C. for 24 hours. Retain dissimilar 
cultures and proceed with the usual routine study. 
Make hanging-drop, stained, and Gram preparations, 
transfer to all media, describe the culture character- 
istics, and make sketches. 

7. Special tests may become necessary after 24 or 
48 hours. Such tests may consist of 



140 LABORATORY GUIDE IN BACTERIOLOGY 

Capsule stain. 

Spore stain. 

Stain for acid-proof bacilli. 

Fermentation tests of all those which produce gas 
in dextrose agar. 

Test for acid in neutral broth. 

Test for agglutination. 

Test for indol. 

Anaerobic cultivation. 

Inoculation of animals. 

For final diagnoses the systematic works of Migula, 
Matzuschita, and Chester should be consulted. 



PART IV 

THE BACTERIOLOGICAL EXAMINATION OF 
WATER AND SEWAGE 



INTRODUCTORY 

This work is designed to follow the physical, chemi- 
cal, and microscopic examination of water. The 
physical examination usually applies to odor, color, and 
turbidity. The chemical examination determines the 
oxygen consumed, dissolved oxygen, free and albumi- 
noid ammonia, nitrites, nitrates chlorin, and hardness. 
The microscopic examination refers to algae, protozoa, 
etc. Many factors influence the results obtained by 
any of these examinations, and in order to gain a clear 
insight into existing conditions judgment on the quality 
of water should not be passed unless all these examina- 
tions and a bacteriological examination have been 
completed. 
References 

Savage, The Bacteriological Examination of Water Supplies, 

London, 1906. 
Horrocks, The Bacteriological Examination of Water, London, 

1901. 
Prescott and Winslow, Elements of Water Bacteriology, New 

York, 1914. 
Ohlmiiller and Spitta, Die Untersuchung and Beurteilung 

des Wassers und Abwassers, Berlin, 1910. 
Kinnicut, Winslow, and Pratt, Sewage Disposal, New York, 

1910. 
Hazen, The Filtration of Public Water Works, New York, 

1900. 

Standard Methods for the Examination of Water and Sewage . 
American Public Health Association, 755 Boylston 
Street, Boston, Mass. 

Bacteriological Standards for Drinking Water. Reprint 
No. 232 from the Public Health Reports, 1914, Wash- 
ington, D.C., Government Printing Office, 1914. 

143 



144 LABORATORY GUIDE IN BACTERIOLOGY 

VVhipple, The Microscopy of Drinking Water, New York f 

John Wiley & Sons, 1914. 

Jackson, Biological Studies by the Pupils of W. T. Sedgwick, 
1906, p. 292. 

Fuller and Johnson, Jour. Exper. Med., 1899, 4, p. 610. 

Don, Chrsholm, Modern Methods of Water Purification, Lon- 
don, 1911. 

Apparatus needed in addition to the list given on 

IX 5- 

Two hundred culture tubes. 

Four wire baskets. 

Twenty fermentation tubes. 

Twelve Erlenmeyer flasks, about 150 c.c. each. 

Six wide-mouth glass-stoppered bottles of about 125 c.c. 
capacity. 

Twenty petri dishes. 

Twenty-five i c.c. pipettes. 

Ten 10 c.c. pipettes. 



SECTION i 

PREPARATION OF CULTURE MEDIA AND OF DILU- 
TION FLASKS 

Dextrose or lactose agar 50 tubes. Thirty-five 
tubes should contain 10 c.c. of agar for plating; the 
others about 7 c.c. 

Nutrient gelatin 20 tubes. This gelatin should 
contain 12 per cent gelatin. This percentage is 
reduced to 10 per cent after the contents of a tube has 
been mixed with the litmus solution and the water used 
for plating if 10 c.c. gelatin are filled into each tube. 

Litmus solution 20 tubes. 

Lactose bile fermentation tubes 30. 



EXAMINATION OF WATER AND SEWAGE 145 

Dilution flasks, filled with 100 c.c. filtered tap water 
or better 100 c.c. o . 8 per cent NaCl solution. After 
sterilization in the autoclave these flasks are assumed 
to contain 99 c.c. each. 

Sugar-free meat infusion broth in bulk for fermenta- 
tion tubes and in tubes 20. 

In addition to these media the media listed on p. 105 
should be prepared. 

The reaction of all media should be adjusted to I 
per cent acid to phenolphthalein. 



SECTION 2 

BACTERIOLOGICAL EXAMINATION OF WATER 
EXERCISE I. COLLECTION OF SAMPLES 

1. Tie a piece of filter paper or lead foil over 6 glass- 
stoppered bottles of about 125 c.c. capacity. Sterilize 
these in the hot-air oven for one hour at 160 C. 

2. Before taking the sample from surface waters 
remove the paper cap, dip the bottle about 12 inches 
below the surface, remove the stopper under water, 
replace the stopper as soon as the bottle is filled, wipe 
dry with a clean cloth or absorbent cotton, and replace 
the paper cap. 

3. Pack the samples in ice in a suitable container. 
They should be kept on ice until ready for examination 
either in the laboratory or in the field. 

4. Samples from pumps should be collected after 
several pails of water have been wasted. 

5. Samples from hydrants should be taken after 
the water has been running freely for at least 15 
minutes. 



146 LABORATORY GUIDE IN BACTERIOLOGY 
EXERCISE 2. EXAMINATION OF SURFACE WATERS 

1. Secure three samples of surface waters from dif- 
ferent sources. 

2. Shake the samples and prepare dilutions. 

Dilution i. 1:10; remove 10 c.c. from a dilution flask con- 
taining loo c.c. sterile water, and replace these 10 c.c. by 10 c.c. 
of the sample. 

Dilution 2. 1:100; add i c.c. of the sample to a dilution 
flask. 

Dilution 3. i : 1,000; add i c.c. of the dilution i : 10 to another 
dilution flask. 

3. Melt a number of dextrose or lactose agar tubes 
in a water bath and cool to 43 C. 

4. Place i c.c. of sterile litmus solution on each of 
12 petri dishes. 

5. Place i c.c. of the sample and i c.c. of each 
dilution on the same petri dishes. 

6. Pour the contents of a tube of the liquefied agai 
on each of the petri dishes and mix. 

7. After the agar has solidified incubate at 37 C. 

NOTE. If working in pairs, it will be instructive to have one 
student incubate the agar plates at 37 C., and the other student 
at room temperature. The period of incubation at 37 C. is 48 
hours, at room temperature 72 hours. It is also instructive to 
plate the same samples and dilutions in gelatin, incubating these 
at 20 C. for 48 hours, and comparing the number of colonies 
with those appearing on agar. 

8. After the plates have been removed from the 
incubator count the colonies, using a colony counter. 
Make differential counts of acid-forming colonies, 
recognized by the reddening of the litmus, and non- 
acid-forming colonies. 



EXAMINATION OF WATER AND SEWAGE 147 

EXERCISE 3. QUALITATIVE AND QUANTITATIVE DE- 
TERMINATION OF B. COLI AND STREPTOCOCCI 

1. Take one sample and inoculate 10 fermentation 
tubes, containing dextrose or lactose broth, each with 
i c.c. of the sample. Similarly inoculate 10 fermenta- 
tion tubes with i c.c. of each of the dilutions. 

NOTE. This experiment requires 40 fermentation tubes and 
may be divided among several students. This same experiment 
should be repeated with lactose bile in place of dextrose or lactose 
broth and the results compared. 

2. Incubate the fermentation tubes at 37 C. 

3. Measure the amount of gas in the closed arm after 
24 hours and replace in the thermostat. 

4. Measure the gas again after 48 hours and deter- 
mine the composition. See p. 81 for directions. 

5. Make smears of each tube and apply Gram's 
stain to determine the presence of streptococci. 

6. Calculate the number of B. coli and streptococci 
present according to the formula given on p. 86. Sub- 
stitute the number of tubes containing streptococci 
for the letter N in the formula to determine the num- 
ber of streptococci. 

NOTE. Each fermentation tube containing gas in the closed 
arm shows the presence of B. coli. On the assumption that at 
least one of the organisms is present in the tube the approximate 
number in i c.c. of water is calculated. 

EXERCISE 4. EXAMINATION OF WELL WATERS 

Secure three samples of well water, make dilutions 
of i : 10 and i : 100 and examine in the same manner as 
directed in Exercises 2 and 3. Compare the results 
with those obtained in Exercise 3. 



148 LABORATORY GUIDE IN BACTERIOLOGY 

EXERCISE 5. EXAMINATION OF RAIN WATER, SNOW, 
AND ICE 

Secure samples of rain water or snow, and of ice. 
Examine these samples, making dilutions up to i : i.ooo 
in the manner directed. 



SECTION 3 
EXAMINATION OF SEWAGE 

1. Secure two samples of sewage. Place one of 
these in an ice chest. Examine the other sample in 
the same manner as directed for water, with this 
exception, that dilutions of 1:1,000, i: 10,000, 1:100,- 
ooo, and 1:1,000,000 are to be used for plating and 
inoculation of fermentation tubes, omitting the lower 
dilutions. 

2. Keep the first sample of sewage in your locker. 

3. After seven days examine both samples, i.e., the 
one kept in the locker and the one kept in the ice chest, 
in the same manner as the fresh sewage was examined. 
Note the difference in the results of this examination 
as compared to that of fresh sewage. 

4. Replace the samples in the locker and ice chest 
and examine again after seven and after 14 days. 

NOTE. If possible a chemical examination for free and albu- 
minoid ammonia and for nitrites and nitrates should be made 
simultaneously with the bacteriological examination. This will 
help to give a clear understanding of the changes which have 
taken place during the three weeks. 



EXAMINATION OF WATER AND SEWAGE 149 

SECTION 4 
DETERMINATION OF ANAEROBES IN SEWAGE 

1. Prepare dilutions of sewage, 1:100, 1:1,000, 
and i : 10,000. 

2. Inoculate a series of 10 litmus milk tubes for 
each dilution with i c.c. of each dilution. 

3. Heat these tubes in a water bath at 80 C. for 
15 minutes. 

4. Incubate at 37 C. for 48 hours, in an anaerobic 
jar (seep. 137). 

5. Determine the number of anaerobes present. 
NOTE. Heating the tubes at 80 C. kills all vegetative forms 

of bacteria, leaving only the spores alive. These develop the 
thermostat and their presence is recognized by violent gas forma- 
tion and breaking -up of the milk curd. The number of anaerobes 
is determined by the same formula as the number of B. coli, sub- 
stituting the number of milk tubes showing growth of anaerobes 
for the letter N in the formula. 



SECTION 5 
STUDY OF B. COLI AND STREPTOCOCCI 

Isolate colonies of B. coli and Streptococcus by plat- 
ing from the fermentation tubes in lactose litmus agar. 
Carry the two organisms through all the media and 
determine whether their characteristics are typical 
according to the rules laid down in the Committee 
Report of the American Public Health Association. 
If they are not typical, they must be rejuvenated 
according to Fuller and Johnson's method. This 



I$o LABORATORY GUIDE IN BACTERIOLOGY 

consists in transferring the cultures for three consecu- 
tive days in broth and incubating at 20 C. From the 
third transfer they are plated in gelatin and a colony 
from this transferred to slant agar, from which sub- 
cultures are made. 



SECTION 6 
ISOLATION OF B. TYPHOSUS FROM WATER 

1. Prepare 10 tubes each of Endo's medium, Dri- 
galski and Conradi's medium, aesc. bile-salt agar (p. 33). 

2. Prepare plates from water containing B. typhosus 
from both of these media. 

3. Incubate at 37 C. for 24 hours and isolate the 
colonies. 

4. Carry the colonies through all media and apply 
the agglutination test with typhoid immune serum. 

SECTION 7 

STUDY OF REACTION OF BACTERIA ON NEUTRAL- 
RED BROTH 

Prepare a number of fermentation tubes with dex- 
trose or lactose broth and add enough of a i per cent 
neutral -red solution until a decided red color has been 
imparted. Inoculate these tubes with a variety of 
colonies from water or sewage plates and note the differ- 
ence in color reaction after 24 and 48 hours' incubation. 



PART V 

THE BACTERIOLOGICAL EXAMINATION OF 
MILK 



INTRODUCTORY 

This work is designed to follow the physical and 
chemical examination of milk. The physical examina- 
tion usually consists in the determination of the specific 
gravity, sediment, odor, and general appearance. The 
chemical examination determines the fat percentage, 
total nitrogen, casein, albumin, milk sugar, acidity, 
total solids, solids not fat, and preservatives, chiefly 
formalin. To determine the exact character of milk, 
physical, chemical, and bacteriological examinations 
should be made. 

References 

Farrington and Woll, Testing of Milk and Its Products, 

Madison, 1908. 
Russell and Hastings, Outlines of Dairy Bacteriology, tenth 

edition, Madison, Wis., H.L. Russell, 1914. 
Ward, Pure Milk and the Public Health, Ithaca, 1909. 
Hygienic Laboratory Bulletin No. 56, "Milk and Its Relation 

to the Public Health, Washington, 1909. 
"Report of the Committee on Standard Methods of the 
Bacterial Analysis of Milk," American Journal of Public 

Health, 755 Boylston Street, Boston, Mass. 
Lafar, Handbuch der technischen Mykologie, Jena, 1905 to 

1908. 

Additional apparatus needed. The outfit given for 
the bacteriological examination of water (p. 143) is 
to be used in this work, with the addition of ten 5 c.c. 
pipettes. 



154 LABORATORY GUIDE IN BACTERIOLOGY 



SECTION i 

PREPARATION OF CULTURE MEDIA AND OF DILU- 
TION FLASKS 

The same culture media will be used as in the bac- 
teriological examination of water (see p. 144), with 
this difference, that dextrose agar only is to be prepared, 
instead of lactose agar. In addition about ten tubes 
of whey agar are to be prepared. 



SECTION 2 

QUANTITATIVE BACTERIOLOGICAL EXAMINATION 
OF MILK 

1 . Secure samples of raw market milk, of pasteurized 
market milk, of certified milk, and of cream. 

2. Prepare dilutions as follows: 

Raw market milk, i : 1,000 and i : 10,000. 
Pasteurized market milk, i : 100 and i : 1,000. 
Certified milk, i : 100. 
Cream, i : 1,000 and i : 10,000. 

3. Plate these in dextrose litmus agar, making 
duplicate plates from each dilution. 

4. Incubate one set at 37 C, the other set at 20 C. 
The incubation period at 37 C. is 48 hours, at 20 C. 
72 hours. 

5. Count the colonies and express the results in 
numbers of bacteria per cubic centimeter of milk. 

6. Make a differential count of acid-forming colonies 
and non-acid-forming colonies. 



EXAMINATION OF MILK 155 



SECTION 3 

EXAMINATION OF MARKET MILK FOR TUBERCLE 
BACILLI 

1. Secure a sample of milk and centrifugalize for 
30 minutes at a speed of about 1,200 revolutions per 
minute. 

2. Mix the sediment and the cream in a sterile tube; 
if too thick add a sufficient amount of sterile o . 8 NaCl 
solution. 

3. Make stains of the mixture for acid-proof bacilli. 

4. Divide the mixture into three parts and inject 
subcutaneously into three guinea-pigs. 

Three weeks is about the shortest time in which 
tuberculosis will develop. The guinea-pigs should be 
watched closely after this time, and if any of them die, 
postmortem examinations of the lesions should be 
made and smears from the affected organs should be 
stained to demonstrate the presence of tubercle bacilli. 
Three guinea-pigs are used for an experiment of this 
nature, because it is impossible to avoid injecting other 
bacteria present in milk, which may cause the death 
of one or more of the guinea-pigs before tuberculosis 
has developed. 

SECTION 4 
A STUDY OF THE ACID FERMENTATION OF MILK 

1. Secure three samples of milk, one of raw milk, 
one of pasteurized milk, and one of certified milk. 

2. Divide each sample into three parts, and keep one 



I5 6 LABORATORY GUIDE IN BACTERIOLOGY 

set in an ice chest, one set in the locker, and the third 
set in a thermostat at 37 C. 

3. Prepare plates in dextrose litmus agar from the 
original three samples and incubate these at 37 C. 

4. Remove every day, with a sterile 5 c.c. pipette, 
5 c.c. of the milk from all samples, determine the 
acidity by titration with 1/20 N'NaOH and phenol- 
phthalein as indicator. When the acidity becomes 
constant titrations may be omitted. 

5. Prepare plates from all samples every day for 
one week, or until the numbers do not increase materi- 
ally. It will be necessary to carry the dilutions up to 
a million after a day or two of market milk, after three 
or four days of pasteurized milk, and toward the end of 
the week of certified milk. 

6. Differential counts of acid-forming and non-acid- 
forming bacteria should be made when this is possible. 

7. After the week has passed, allow the samples 
to stand for two weeks longer and make titrations and 
plates every three days until the acidity and the num- 
ber of colonies are constant. 

The results of this study should be tabulated, as 
they will illustrate the process of "natural souring" 
of milk. 

If milk is kept at 37 C. for three weeks or longer the 
acidity often reaches more than 2 per cent and even 
up to 3 per cent. This is due to the activity of a group 
of bacteria wholly different from the ordinary lactic 
acid bacteria. These bacteria grow poorly on ordi- 
nary media. Their presence may be demonstrated by 
plating the sour milk in beerwort agar, or better, in 
whey agar (see p. 36). 



EXAMINATION OF MILK I$7. 



SECTION 5 

DETERMINATION OF B. COLI AND STREPTOCOCCI IN 
MILK 

i. Inoculate a series of ten dextrose broth fermen- 
tation tubes with i c.c. of market milk diluted 1:10, 
another series of ten fermentation tubes with a dilution 
of 1:100, another series 1:1,000. 

2. Incubate these tubes at 37 C. 

3. Measure the gas formed after 24 and 48 hours 
and determine the composition of the gas after 48 
hours. Directions for this work are given on p. 81. 

4. Determine the number of B. coli present in i c.c. 
of the milk according to the formula on p. 82. 

5. Make smears from each fermentation tube and 
stain with Gram stain. 

6. Examine these smears for streptococci and 
determine the number according to the same formula, 
substituting the number of tubes containing strepto- 
cocci for the letter N in the formula. 

7. Plate out one of the fermentation tubes contain- 
ing both B. coli and streptococci and carry some of the 
colonies through all media. 

NOTE. The work of this exercise should be repeated with 
pasteurized and certified milk. For pasteurized milk the undi- 
luted sample and dilutions of i : 10 and 1:100 should be used, for 
certified milk the undiluted sample and a dilution of i : 10. 

Estimation of numbers of B. coli in milk may be made by 
inoculation of fermentation tubes containing dextrose broth 
with falling amounts of milk, i.e.. i c.c.. o. i c.c.. o.oi c.c., etc. 



I $8 LABORATORY GUIDE IN BACTERIOLOGY 



SECTION 6 

A COMPARATIVE STUDY OF THE EFFECTS OF PAS- 
TEURIZATION AND SO-CALLED STERILIZA- 
TION OF MILK 1 

. i. Mix three quarts of raw milk and pour plates 
to determine the number of bacteria and the number 
of acid-forming bacteria. 

2. Divide the mixed milk into three parts. Heat 
one-third for 20 minutes at 65 C., boil one- third for 
several minutes, and place all parts in an ice chest. 

3. As soon as cooled, prepare plates of the milk 
heated to 65 C. (pasteurized) and of the boiled milk. 
Note the "cooked" taste and odor of the boiled milk. 

4. Check the keeping qualities of the three kinds of 
milk by preparing plates on three successive days, 
and after that every other day for six days. 

It is of importance to make differential counts and 
determine the ratio of acid-forming and non-acid- 
forming bacteria in all plates. 



SECTION 7 

A QUALITATIVE AND QUANTITATIVE STUDY OF 
ANAEROBES IN MILK 

1. Secure a sample of raw milk, one of pasteurized 
milk, and one of certified milk. 

2. Prepare dilutions of the raw milk i : 10, i : 100, and 

1 The term " sterilized milk " is commonly used for boiled milk. 
In a bacteriological sense boiled milk is not always sterile, some 
of the spores being able to survive boiling. 



EXAMINATION OF MILK 159 

i : 1,000; of the pasteurized milk mo and 1:100, and 
of the certified milk i : 10. 

3. Inoculate a series of 10 litmus milk tubes with 
i c.c. each of the raw milk, a series of 10 milk tubes 
with i c.c. each of the dilution i : 10, and the same with 
the other dilutions and the other milks. 

4. Heat these tubes in a water bath to 80 C. for 
15 minutes. 

5. Incubate anaerobically for 48 hours at 37 C. 

6. Anaerobes will give evidence of their presence 
by the breaking-up of the curd formed, and by violent 
evolution of gas. The number may be determined 
according to the formula given on p. 86 by substitut- 
ing the number of milk tubes with growth for the 
letter N in the formula. 



SECTION 8 

A STUDY OF SOME ORGANISMS CAUSING ABNORMAL 
FERMENTATIONS IN MILK 

1. Place about 50 c.c. certified milk in each of 5 
Erlenmeyer flasks. 

2. Sterilize these flasks in the autoclave for 5 
minutes. 

3. Inoculate these flasks with laboratory cultures of 
B. prodigiosus, B. cyanogenes, B. viscosus, Sarcina 
lutea, and Torula amara. 

4. Allow these to remain in the locker or in an 
incubator at 20 C. for three days; examine the con- 
ditions as to color and consistency. 



160 LABORATORY GUIDE IN BACTERIOLOGY 



SECTION 9 

EXAMINATION OF MILK FOR MOLDS AND 
YEASTS 

1. Prepare plates in beerwort agar from undiluted 
milk and from dilutions of i : 10, and i : 100. 

2. Incubate these at 37 C. and examine for molds 
and yeasts. 



SECTION 10 
EXAMINATION FOR LEUKOCYTES IN MILK 

Examination for leukocytes in milk is carried on in 
many laboratories and the test was formerly considered 
of much value. Recent work, however, has cast 
doubt on its significance, since it has been found that 
milk of unquestioned purity and soundness may contain 
as high as 1,000,000 leukocytes per cubic centimeter, 
and with modern methods it is not possible to distin- 
guish between leukocytes and pus cells. Leukocytes 
are always present in milk in variable numbers. Some 
observers believe that the presence of leukocytes and 
streptococci together in large numbers indicate pus 
in the milk. The reliability of this test, however, has 
not been sufficiently demonstrated. 

EXERCISE i. STOKES' METHOD 

1. Centrifugalize 100 c.c. of milk. 

2. Smear the sediment on a cover glass. 

3. Examine under the oil-immersion lens and record 
the number of leukocytes and streptococci. 



EXAMINATION OF MILK 161 

More than five cells in a field are sufficient to con- 
demn the milk. 

EXERCISE 2. STEWART'S METHOD, MODIFIED BY SLACK 

1. Fill 2 c.c. of milk into a tube provided for the 
purpose. 

2. Centrifugalize at a speed of 2,500 to 3,000 revo- 
lutions a minute. 

3. The stopper is then removed and the sediment 
smeared on a slide so as to cover 4 square centimeters. 

4. Examine the slide under the oil-immersion lens 
and count the cells and streptococci. 

More than 50 cells are sufficient to condemn the 
milk. 

EXERCISE 3. DOANE-BUCKLEY METHOD, MODIFIED BY 
RUSSELL AND HOFFMANN 

1. Heat sample of milk for i minute at 85 C. 

2. Centrifugalize 10 c.c. for 20 minutes. 

3. Remove cream and milk, leaving o . 5 c.c. milk. 

4. Mix and place in a blood counter. 

5. Allow to settle for 2 minutes. 

6. Count the cells in several hundred squares. 
The average number of cells per square is multiplied 
by 200,000 to arrive at number of cells per c.c. of milk. 

EXERCISE 4. PRESCOTT AND BREED'S METHOD 

1. Remove with capillary pipette with rubber bulb 
one drop (o.oi c.c.) of the shaken sample of milk. 

2. Spread over i square centimeter of a glass slide. 

3. Dry by gentle heat. 

4. Dissolve out the fat with xylol. 

5. Fix smear in alcohol for a few minutes. 

6. Overstain with methylene blue. 

7. Decolorize with alcohol. 



162 LABORATORY GUIDE IN BACTERIOLOGY 

SECTION II 

A STUDY OF GROUPS OF BACTERIA IN MILK 
(Ayers and Johnson) 

1. Prepare plates from a sample of milk. 

2. Pick off all colonies from a suitable plate and 
inoculate each colony in a tube of litmus milk. 

3. Incubate for 2 weeks at 37 C. 

4. Note all reactions and classify in groups as 
follows: 

a) Acid-coagulating group. 

b) Acid-forming without coagulation. 

c) Inert group, i.e., no change in the milk. 
<f) Alkali-forming group. 

e) Peptonizing group. 

This experiment should be made with raw milk and 
the same milk pasteurized. The results should be 
compared. 



PART VI 

THE BACTERIOLOGICAL EXAMINATION OF 
SOIL 



INTRODUCTORY 

For this work a knowledge of elementary bacteri- 
ology and quantitative and qualitative chemistry is 
indispensable. 

References 

Lafar, Handbuch der technischen Mykologie, Jena, 1905 to 

1908. 
Bulletin 94, Iowa Experiment Station (Soil Samplers). 

Apparatus needed in addition to the list given on 

P. 5: 

Two hundred culture tubes. 

Twelve Erlenmeyer flasks, 150 c.c. each. 

Six Erlenmeyer flasks, 500 c.c. each. 

Twelve fermentation tubes. 

One mortar and pestle (wedgewood or porcelain). 

One soil sampler. 

The media required may be made up as the work 
requiring them comes up. Besides the special media 
which will be needed a set of media similar to those 
given for medical bacteriology will be used. See p. 105, 



SECTION i 

QUANTITATIVE DETERMINATION OF BACTERIA AND 
SPORES IN SOIL 

EXERCISE I. SECURING SAMPLES OF SOIL 

Soil samplers are used and cylinders of soil removed 
from the upper eight inches of soil. Cylinders of soil 
should be taken in various parts of the field so as 
165 



166 LABORATORY GUIDE IN BACTERIOLOGY 

to obtain an average representation of the soil. The 
cylinders are wrapped in sterile paper provided for 
that purpose and taken to the laboratory. Take 
samples in this manner from various kinds of soil, viz., 
garden soil, sand, loam, clay, manured fields, etc. 

EXERCISE 2. PREPARATION OF SAMPLES FOR BAC- 
TERIOLOGICAL EXAMINATION 

1. The samples from the same kind of soil are mixed 
in a sterile mortar. 

2. Place part of each of these composite samples 
in a sterile glass tube. 

3. Weigh accurately 10 grams from each composite 
sample and dry in a hot-air oven for one hour at 100 C. 
Cool and place in a desiccator. After 24 hours weigh 
again and calculate the percentage of dry matter 
present. 

EXERCISE 3. PREPARING SOIL SUSPENSIONS AND 
DILUTIONS AND POURING PLATES 

1. Prepare .sterile dilution flasks. Fill 100 c.c. 
water in Erlenmeyer flasks of about 150 c.c. capacity. 
Sterilize these in the autoclave for 10 minutes. As 
there is a loss by evaporation during the process of 
sterilization the remaining amount is approximately 
99 c.c. 

2. Weigh the tube containing the sample of soil. 

3. Remove about 10 grams by shaking the tube or 
by means of a sterile knife or spatula. Place in a 
sterile mortar. 

4. Weigh the tube again and determine the precise 
amount of soil removed. 

5. Grind the soil sample in the mortar with some 



EXAMINATION OF SOIL 167 

sterile water from one of the dilution flasks for one 
minute. 

6. Add some more water, so that the final volume 
is 100 c.c. The suspension should be perfectly uniform. 

7. Shake the suspension vigorously for three 
minutes. 

8. Prepare dilutions from this suspension in the fol- 
lowing manner: Add i c.c. suspension to a flask with 
99 c.c. sterile water. Shake well. Remove 10 c.c. 
water from another flask and add 10 c.c. of the dilu- 
tion of i : 100 to this flask. This makes a dilution of 
i : 1,000. Then add i c.c. of the dilution of 1:100 to 
another flask, making a dilution of i : 10,000. i c.c. 
from the dilution i : 1,000 added to another flask makes 
a dilution of i : 100,000. Finally add i c.c. of the dilu- 
tion i : 10,000 to another flask, making a dilution of 
1:1,000,000. 

9. Pour duplicate agar plates from all dilutions and 
incubate for four days at room temperature. Plates 
should be prepared in both agar and gelatin. 

10. Examine the gelatin plates daily, and count the 
colonies earlier than four days if there are many 
liquefiers present. 

11. After counting the colonies on both agar and 
gelatin calculate the number present in one gram of 
dry soil. 

12. Examine the colonies and determine as near as 
possible the group to which they belong according to 
Chester's Determinative Bacteriology. In most cases 
stained preparations and hanging drops will give this 
information. If not, cultures on slant agar must be 
prepared and the organisms studied in the usual 
manner by making subcultures. 



168 LABORATORY GUIDE IN BACTERIOLOGY 

EXERCISE 4. DETERMINATION OF THE* NUMBER OF 
SPORES IN SOIL 

1. Add i c.c. of the soil suspension 1:1,000 to a 
tube of melted gelatin. 

2. Heat the tube in a water bath to 80 C. for 10 
minutes. 

3. Pour the gelatin on a petri dish and incubate 
at room temperature for three or four days. 

4. Count the colonies and calculate the number 
for one gram dry soil. 

Each colony represents one spore originally present 
in the soil. 



SECTION 2 

A STUDY OF THE PEPTONIZATION OF PROTEINS BY 
SOIL BACTERIA 

EXERCISE I. DIGESTION OF BLOOD SERUM 

1. Place 200 c.c. blood serum in a 500 c.c. flask 
and sterilize in the autoclave. 

2. After cooling, add 5 c.c. of a suspension of garden 
soil in water. 

3. Incubate at 37 C. until all of the serum has been 
liquefied. 

EXERCISE 2. DETERMINATION OF THE CHANGES PRO- 
DUCED IN BLOOD SERUM 

1. Add a suitable quantity of distilled water to the 
liquefied blood serum. 

2. Filter to remove bacterial masses and impurities. 

3. Neutralize with NaOH, using litmus as indi- 
cator. 



EXAMINATION OF SOIL 169 

4. Boil the solution. If a precipitate forms, it is 
due to undigested albumins. 

5. Filter and add ammonium sulphate to the nitrate 
to saturation. Albumoses will be precipitated. 

6. Allow the mixture to stand over night, then 
filter. 

7. Dissolve the precipitate in distilled water. 

8. Add to the dissolved precipitate an excess of 
NaOH and a few drops of copper sulphate solution. 
This will produce the characteristic color of the biuret 
reaction for the albumoses present. 

9. Add to the nitrate left from No. 6 an excess of 
NaOH and a few drops of copper sulphate solution. 
This will give the biuret reaction for peptons. 

EXERCISE 3. THE DIGESTION OF GELATIN 

1. Prepare gelatin plates with a dilution of garden 
soil suspension. 

2. Isolate one of the liquefying colonies by trans- 
ferring it to slant agar. Make subcultures and 
determine the species. 

3. Sterilize 200 grams of gelatin in a 500 c.c. flask 
and inoculate this heavily with a culture of the lique- 
fying organism obtained in the previous experiment. 

4. Incubate at room temperature until the gelatin 
is completely liquefied. 

5. Determine the composition in the same manner 
as in Exercise i. 

EXERCISE 4. THE DIGESTION OF CASEIN 

1. Precipitate the casein in a quantity of milk by 
means of dilute acetic acid. 

2. Filter. 



170 LABORATORY GUIDE IN BACTERIOLOGY 

3. Wash the precipitate with dilute acetic acid until 
the milk sugar has been removed. 

4. Place in a 500 c.c. flask and add 300 c.c. distilled 
water. 

5. Neutralize with NaOH and sterilize in the auto- 
clave. 

6. Inoculate with soil suspension. 

7. Incubate at 37 C. until all the casein has been 
dissolved. 

8. Determine the composition of the solution as in 
Exercise i. 

9. Make plates in gelatin and isolate the organisms. 
NOTE. The precipitated casein has to be washed in order 

to remove all traces of milk sugar, otherwise acid-forming bacteria 
will multiply rapidly and crowd liquefiers out. 



SECTION 3 

THE FORMATION OF AMIDO COMPOUNDS AND 
AMMONIA 

EXERCISE I. THE FORMATION OF INDOL 

1. Prepare 200 c.c. Dunham's solution and place 
in a 500 c.c. flask. 

2. Sterilize in the autoclave. 

3. Inoculate with a pinch of garden earth and incu- 
bate at 37 C. for one week. 

4. Remove a small amount with a sterile pipette and 
add a few drops sulphuric acid. Follow this up with 
a few drops of a o.ooi per cent solution of potassium 
or sodium nitrite. 

The appearance of a red color indicates the presence 
of indol. 



EXAMINATION OF SOIL 171 

EXERCISE 2. THE FORMATION OF AMMONIA 

Experiment i. Formation of ammonia from pep- 
ton 

1. Test the remainder of the solution in Exercise i 
for ammonia. 

2. Make a quantitative determination of ammonia 
by distillation and nesslerization. 

Experiment 2. Formation of ammonia from urea 

1. Prepare a solution as follows: 

Urea i gram 

Pepton 0.2 gram 

Distilled water 100 c.c. 

This solution must not be sterilized, as heat breaks 
up urea into ammonia and carbon dioxid. 

2. Inoculate with some garden earth. 

3. Incubate at room temperature for one week. 

4. From time to time moisten a piece of filter paper 
with Nessler's solution and pour a few drops from the 
flask on the paper. A yellow or brown color indicates 
the formation of ammonia. 



SECTION 4 

THE FORMATION OF NITRITES FROM AMMONIA 
AND ISOLATION OF NITRITE BACTERIA 

EXERCISE I. THE FORMATION OF NITRITES 

1. Prepare five flasks and place 50 c.c. of the solu- 
tion on p. 38 in each. Number these flasks con- 
secutively. 

2. Inoculate flask i with soil, and incubate at room 
temperature. 



172 LABORATORY GUIDE IN BACTERIOLOGY 

3. As soon as growth has appeared transfer some of 
the growth from the first flask to the second. 

4. As soon as growth has appeared in the second 
flask transfer some of the growth to the third flask. 

5. Repeat the procedure until all five flasks have 
been inoculated. This method of transferring repeat- 
edly gradually weeds out all bacteria but those which 
are able to grow in the solution, and these may thus 
be obtained in pure culture. 

6. Examine these bacteria by making stains, hang- 
ing drop, spore stain, etc. 

7. Test each flask for nitrites with the starch iodin 
test as follows. Prepare the following solutions: 

a) Dissolve 2 grams starch in 100 c.c. of water by 
boiling. 

b) Dissolve one gram potassium iodid in 100 c.c. of 
distilled water. 

The potassium iodid solution must be freshly pre- 
pared. Add a few drops of diluted H 2 SO 4 to liberate 
nitrous acid. Then add i c.c. of each of the solutions 
to 5 c.c. of the fluid to be tested. If nitrous acid is 
present, iodin will be liberated and will give a blue 
reaction with starch. 

EXERCISE 2. ISOLATION OF NITRITE BACTERIA 

Experiment i. Agar method 

1. Prepare the agar medium described on p. 38. 

2. Fill 10 tubes, and sterilize in the autoclave. 

3. Pour plates from this medium inoculated with 
the contents of the flasks prepared in Exercise i in 
various dilutions. 

4. Incubate at room temperature until growth has 
appeared. 



EXAMINATION OF SOIL 173 

5. Transfer some of the colonies to slanted agar 
prepared as described on p. 38. 

6. Study the colonies by stains, hanging drop, etc., 
and identify the organism. 

Experiment 2. Silica jelly method 

1. Prepare some tubes with silica jelly as described 
on p. 38. 

2. Melt these tubes, inoculate several with a soil 
suspension, others with the contents of the flasks 
prepared in Exercise i, others with colonies obtained 
by the washed agar method. 

3. Incubate the plates at room temperature and 
study the colonies in the usual manner. 

. Collodion sacs for the dialysis of silica are made in 
the following manner: 

Depending on the size desired a culture tube or an 
Erlenmeyer flask or any other suitable glass vessel can 
be used. The finished sac is always much smaller 
than the vessel used and this shrinkage must be con- 
sidered in choosing the right size of vessel. Celloidin 
is dissolved in a mixture consisting half of ether and 
half of alcohol in such quantity that the solution con- 
tains 6 per cent celloidin. Celloidin usually is covered 
with water. This must be washed off with alcohol 
before the celloidin is dissolved. A quantity of the 
celloidin solution is then poured into the flask and the 
flask rotated until the whole inside surface is covered 
with the solution. The flask is then inverted and the 
celloidin allowed to drain off so that a thin coat re- 
mains on the sides of the flask. The flask must then 
be rotated and air blown into it until the celloidin is 
almost dry. The process is then repeated so that the 



174 LABORATORY GUIDE IN BACTERIOLOGY 

inside surface of the flask is covered with two coats. 
For large sacs three coats should be applied. When 
the celloidin is nearly dry water should be run into 
the flask until filled. This water should be changed 
several times. Commencing at the top of the flask 
the film is separated from the glass and by means of a 
smooth instrument, for instance, a glass rod which 
has been rounded off at the end, the film is slowly 
removed from the glass. As soon as there is a fairly 
large part of the film separated, the water may be 
emptied out and water run in between the film and the 
glass. In this manner the film is gradually removed 
from the glass. If the celloidin is too dry before the 
flask is filled with water, it will crack and will be 
difficult to remove from the glass. The technic is not 
difficult and with some practice and care sacs can be 
made which will be large enough to hold two liters of 
liquid. 

SECTION 5 

THE FORMATION OF NITRATES FROM NITRITES, 
AND ISOLATION OF NITRATE BACTERIA 

EXERCISE I. THE FORMATION OF NITRATES FROM 
NITRITES 

1. Prepare a solution as described on p. 39. 

2. Prepare a series of five flasks and place 50 c.c. of 
the solution in each. 

3. Sterilize in the autoclave. 

4. Inoculate the first flask with soil and incubate at 
room temperature. 

5. As soon as growth has appeared inoculate a 
second flask, from this one a third, etc., as in Exercise i, 



EXAMINATION OF SOIL 175 

Section 4. The bacteria will finally appear in pure 
culture. 

6. Determine the presence of nitrates. For this 
determination proceed as follows: 

a) Preparation of phenol sulphonic acid: 30 grams 
phenol are dissolved in 370 grams concentrated 
sulphuric acid in a round-bottom flask. Immerse 
completely in a water bath and heat for six hours. 

b) Dissolve 20 grams KOH in 20 c.c. distilled water. 

c) Evaporate a small amount of the culture to dry- 
ness on a water bath. 

d) Add to the residue one c.c. phenol sulphonic 
acid and rub with a glass rod. 

e) Add enough KOH solution to make the solution 
alkaline. 

/) Dilute with distilled water. 
If nitrates are present this will be indicated by the 
appearance of a yellow color. 

EXERCISE 2. ISOLATION OF NITRATE BACTERIA 

These -bacteria may be isolated with agar or silica 
jelly in the same manner as in Section 4, Exercise 2. 

SECTION 6 

THE ASSIMILATION OF FREE ATMOSPHERIC NITRO- 
GEN AND ISOLATION OF THE BACTERIA 

EXERCISE I. BY LEGUME BACTERIA (TUBERCLE 

BACTERIA) 

Experiment i 

i. Secure the roots of a clover plant, or any other 
legume. Note the appearance and distribution of 
the tubercles. 



176 LABORATORY GUIDE IN BACTERIOLOGY 

2. Crush between two slides a small tubercle and a 
large one. 

3. Make mounted preparations and examine them 
under the microscope. 

Experiment 2 

1. Wash a number of tubercles in distilled water, 
then soak in mercuric chlorid solution 1:1,000 to 
sterilize the exterior of the tubercles. 

2. Wash again in several changes of sterilized 
distilled water. 

3. Crush in a sterile petri dish. 

4. Plate in agar, prepared as described on p. 38. 

5. Incubate at room temperature. 

6. After growth has appeared transfer to all ordinary 
laboratory media. 

7. Study characteristics in the usual manner. 



EXERCISE 2. BY ORGANISMS OTHER THAN LEGUME 
BACTERIA 

Experiment i 

1. Prepare 5 flasks and place 50 c.c. of the solution 
on p. 39 in each flask. 

2. Sterilize in the autoclave. 

3. After growth has appeared inoculate a second 
flask from the first, from the second to the third, etc. 

4. Determine the gain in nitrogen by the Kjeldahl 
method. This determination should be repeated at 
regular intervals to determine the progress of nitrogen 
assimilation. 

Experiment 2. These organisms may be obtained 
in pure culture by plating in agar or silica jelly media. 



EXAMINATION OF SOIL 177 



SECTION 7 

THE REDUCTION OF NITRATES TO NITRITES AND 
ISOLATION OF THE BACTERIA 

1. Prepare a flask of pepton broth and add 0.5 per 
cent potassium nitrate. 

2. Inoculate with a small quantity of soil. 

3. Incubate for several days at room temperature. 

4. Test by the starch iodin method as described in 
Section 4, Exercise i. 

5. Obtain pure cultures by plating on nutrient 
agar. 



SECTION 8 
THE REDUCTION OF NITRATES TO FREE NITROGEN 

1. Prepare several large fermentation tubes with 
the medium described on p. 40. 

2. Inoculate the closed arm of the fermentation tubes 
with varying quantities of soil. 

3. Incubate at 37 C. 

4. When gas evolution has ceased determine the 
percentage of gas. 

5. Determine the composition of the gas. 

6. These bacteria may be isolated by plating in 
agar, or better in a medium prepared by adding agar 
to the original solution used in the fermentation tubes. 

7. Study the morphology of the bacteria in pure 
culture. 



178 LABORATORY GUIDE IN BACTERIOLOGY 



SECTION 9 

GROWING LEGUMES IN SAND AND IN SAND INOCU- 
LATED WITH TUBERCLE BACTERIA 

Secure some flower pots and fill them with sand. 
Plant some species of legume and soak the sand in half 
the number of pots with suspensions of legume bacteria. 
The difference in growth between those planted in pure 
sand and those planted in inoculated sand will be evi- 
dent after a short time. 



PART VII 

MOLDS, YEASTS, TORULAE, AND ACETIC-ACID 
BACTERIA 



INTRODUCTORY 

References 

Klocker, Die Gahrungs Organismen (translated into English). 

Hansen (translated by Miller), Practical Studies on Fermenta- 
tion, London, 1896. 

Jorgensen, Microorganisms and Fermentation, London, 1900. 

Green, Soluble Ferments and Fermentation, Cambridge, 1901. 

Lafar, Handbuch der technischen Mykologie, Jena, 1905 8. 

Marshall, Microbiology, Philadelphia, P. Blakiston, Son & 

Co., 1911. 
Apparatus needed in addition to the list on p. 5: 

Fermentation tubes 24 

Erlenmeyer flasks, 150 c.c. each 24 

Stender dishes 10 

Glass tumblers 10 

SECTION i 
PREPARATION OF CULTURE MEDIA 

Prepare 25 tubes of each of the following media: 

Meat extract agar, partly for slants and partly for 
plating. 

Dextrose agar. 

Litmus milk. 

Liquid beerwort. 

Beerwort agar for plating. 

Beerwort gelatin. 

Also fill 12 Erlenmeyer flasks with 50 c.c. beerwort 
each, and 

Meat extract broth 5 tubes. 

Beerwort may be sterilized in a cask in the follow- 
ing manner: Fit a cork with two holes in the bunghole 

181 



1 82 LABORATORY GUIDE IN BACTERIOLOGY 

and insert two glass tubes through the holes. The 
tubes should reach to the bottom of the cask (see 
Fig. 35). The open ends of both tubes should be 
plugged with cotton and have a constriction below the 
cotton, to prevent it from slipping down. One of the 
tubes is connected with the air valve of an autoclave 
and steam passed through the wort for 30 minutes. The 
process is repeated on the two following days. Sterile 
wort may be obtained from the cask by blowing through 
the tube e and collecting from the bent tube, which, 
when not in use, should be protected from contamina- 
tion by a sterile test tube with a cotton plug. 



SECTION 2 

A STUDY OF MOLDS 

EXERCISE I. COLLECTING MOLDS FROM THE AIR 

1. Expose two plates of wort agar and two of meat 
extract agar to the air in different places. 

2. Incubate at 37 C. 

3. After molds have appeared transfer three different 
colonies to slanted wort agar. Transfers are made by 
touching the ends of the hyphae with a platinum needle 
and streaking on the slanted surface of a tube of wort 
agar. 

4. After full development transfer some of the spores 
to liquid wort or broth. This is done by touching the 
ends of the filaments with a platinum needle. 

5. Examine a large loopful of this spore suspension 
under the microscope, using a low magnification. 



MOLDS, YEASTS, TORULAE, AND BACTERIA 183 

6. If the spores are numerous dilute the suspension 
until only a few appear in a loopful. 

7. When the dilution is high enough to show but 
a few spores under the microscope transfer a large 
loopful to a cover slip. 

: Ji> 




FIG. 34 

Sterilizing Wort in a Cask 

a. Cask b. Rubber stopper c. Cotton plugs d. Test tube 
e. Glass tubes /. Cotton stopper and connection with autoclave 

8. Invert this cover slip over the ring of a Bb'ttcher 
Moist Chamber (Fig. 35). 

9. Incubate at 37 C. 

10. Observe the hanging drop every day and make 
sketches. After 7 to 10 days the cycle of development 
will be completed so that a new crop of spores has 
appeared. 



184 LABORATORY GUIDE IN BACTERIOLOGY 

The Bottcher Moist Chamber consists of a slide, a 
cover slip, and a glass ring (see Fig. 35). The glass 
ring is held on the slide by a mixture of rosin and castor 
oil. A few drops of sterile water are placed in the 
bottom to maintain moisture in the hollow of the ring. 
The inverted cover slip with the hanging drop is held 
in position by smearing vaselin around the top of the 
glass ring. 




FIG. 35 

Bottcher Moist Chamber 

a. Slide ft. Glass ring c. Cover slip 

d. Hanging drop e. Sterile water 

EXERCISE 2 

Preparation of slides for microscopic study of molds, 
also stained preparations (see p. 79). 

EXERCISE 3. STUDY OF MOLDS IN CHEESE 

1. Secure some Roquefort and some Camembert 
cheese. 

2. Pick out with a looped needle some of the dark 
spots in the Roquefort cheese and some of the mold on 
the Camembert cheese. 

3. Make streaks of this material on wort agar plates. 

4. Isolate the molds and study as in Exercises i 
and 2. 

EXERCISE 4. STUDY OF THE AMYLOLYTIC ACTION OF 
MOLDS 

1. Prepare cultures of Aspergillus oryzae. 

2. Add some starch to liquid wort in a flask and 
sterilize. 



MOLDS, YEASTS, TORULAE, AND BACTERIA 185 

3. Inoculate with the mold and test for sugar 
formation by the Fehling test from day to day. 

EXERCISE 5. CULTIVATION OF MOLDS IN 
SOLUTION 

1. Preparation of Raulin's solution (see p. 42). 

2. Place about 50 c.c. of the solution in three Erlen- 
meyer flasks and sterilize in the autoclave. 

3. Inoculate the surface with molds from three 
species of molds. 

4. Incubate at 37 C. 

5. After growth has appeared examine the molds 
and make sketches. 

EXERCISE 6. A STUDY OF MOLDS FROM LABORATORY 
CULTURES 

Study cultures of molds furnished in the same 
manner as outlined in the first five exercises of this 
section. 



SECTION 3 

A STUDY OF YEASTS 

NOTE. All yeast cultures are to be incubated at 25 C. unless 
otherwise directed. 

EXERCISE I. STUDY OF YEASTS FROM THE AIR 

1. Expose two plates of wort agar to the air in 
different places, or if the plates exposed in the previous 
section have colonies of yeasts these may be used. 

2. Transfer three colonies, which appear to differ 
from each other by microscopic examination, to tubes 
of slant wort agar, and incubate. 



186 LABORATORY GUIDE IN BACTERIOLOGY 

3. After growth has taken place transfer from these 
agar slants to the following media: Meat agar slant, 
dextrose agar, litmus milk, wort gelatin, liquid wort. 

4. After 24 hours' incubation make stains with 
gentian violet and by Gram's method, examine the 
unstained cells in water, and make sketches. 

EXERCISE 2. A STUDY OF GAS EVOLUTION BY YEASTS 

1. Prepare 2 per cent solutions of the following 
carbohydrates in wort: Dextrose, saccharose, lactose, 
mannit, levulose, maltose. 

2. Fill three fermentation tubes from each of these 
solutions and sterilize for three consecutive days in the 
Arnold. 

3. Inoculate each set with the three yeast cultures 
studied in Exercise i. 

4. Measure the percentage of gas evolved after 
24 and 48 hours. 

5. Determine approximately the composition of the 
gas formed (see p. 82), using a 4 per cent NaOH 
solution. 

6. Tabulate the results, stating the amount and 
composition of the gas formed from each carbohydrate 
by the three species of yeasts. 

EXERCISE 3. A STUDY OF FILM FORMATION 

1. Prepare cultures in liquid wort from three species 
of yeasts. 

2. After growth has appeared inoculate three flasks 
containing sterile wort with each one of the yeasts. 
This should be done after pouring the supernatant 
liquid off, inoculating with a loop from the sediment. 

3. Incubate these flasks as follows: One set at 3 7 C., 



MOLDS, YEASTS, TORULAE, AND BACTERIA 187 

the second set at room temperature, and the third set 
at 25 C. 

4. Note which temperature is most favorable to 
film formation. 

5. Examine under the microscope some cells from 
the film and some from the sediment, also make per- 
manent stained preparations. It will be observed 
chat there is a difference in morphology between the 
cells from the film and those from the sediment. Also 
note the decolorization of the wort. 

6. Determine by distillation approximately the 
amount of alcohol produced. 

EXERCISE 4. A STUDY OF SPORE FORMATION 

1. Prepare cultures in liquid wort. 

2. Prepare gypsum blocks as described on p. 80. 

3. After growth has taken place in the tubes pour 
off the supernatant fluid and smear the sediment on the 
surface of the gypsum blocks. 

4. Incubate the gypsum blocks at 25 C. 

5. Examine a small amount from day to day until 
spores are present, and make sketches. . 

EXERCISE 5. PREPARATION OF PURE CULTURES OF 
YEASTS FROM ONE CELL 

The preparation of pure cultures from one cell has 
become of vast importance in breweries. It is rela- 
tively difficult to prepare pure cultures of bacteria 
from one cell, these being smaller than yeasts and, 
therefore, more difficult to manipulate. Barber 
(Jour. Infect. Dis., 1908, 5, p. 379) has succeeded in 
devising a practical method for isolating single cells of 
bacteria and preparing pure cultures from these. 



1 88 LABORATORY GUIDE IN BACTERIOLOGY 

Experiment i. The dilution method 

1. Prepare a culture of a species of yeast in liquid 
wort. 

2. Examine a large loopful under the microscope 
and dilute the culture with sterile wort until there is 
an average of about one cell to the loopful. 

3. Inoculate a series of ten tubes of liquid wort 
with a loopful each of the diluted culture, or inoculate 
with a drop from a sterile capillary pipette. 

4. Incubate. 

All those tubes in which growth has appeared con- 
tain a culture originating from one cell. 

Experiment 2. Hansen's gelatin method 

1. Prepare a culture of a mixture of two or three 
species of yeast. 

2. After growth has appeared inoculate a tube with 
liquefied wort gelatin at a temperature of about 30 to 
35 C. 

3. Examine under the microscope and dilute with 
liquefied gelatin until there are only one or two cells 
to each loopful. 

4. Prepare cover slips in the following manner: 
Dip several cover slips into liquid paraffin. After the 
paraffin has solidified draw lines through the paraffin 
with a sharp steel needle, so as to form a number of 
squares. Mark each square with numbers or letters 
with the same needle. Dip the cover glass into hydro- 
fluoric acid for a few seconds, wash in water, then 
chloroform, ether, and alcohol until the glass is clean 
and free from fat. The hydrofluoric acid will have 
etched the glass so as to show the marks permanently. 

5. Cover the slip with a thin coat of the diluted 
wort gelatin containing the culture. 



MOLDS, YEASTS, TORULAE, AND BACTERIA 189 

6. After the gelatin has solidified, paint the top of 
the glass ring of a Bottcher Moist Chamber with 
vaselin and invert the cover slip, gelatin down. Do 
not neglect to place a small amount of water inside the 
moist chamber. 

7. Examine under the microscope and make a 
sketch corresponding to the figures on the cover slip 
and make a mark for all single cells observed. It is 
possible then to find the cells again by comparing the 
cover slip with the sketch. 

8. Incubate at room temperature. 

9. Observe from day to day the growth of colonies 
from single cells. 

10. When the colonies are large enough to be picked 
up with a platinum needle, inoculate several tubes of 
liquid wort with the colonies. 

11. Incubate at 25 C. and after growth has 
appeared transfer the sediment from each tube to a 
flask containing liquid wort. 

12. Incubate these flasks and study the cultures by 
making microscopic examination and stains, study the 
film and the sediment separately, inoculate fermenta- 
tion tubes with the various carbohydrates, and inoculate 
gypsum blocks. Also note the aroma, and determine 
the amount of alcohol formed. 

EXERCISE 6. A STUDY OF LABORATORY CULTURES 

Prepare cultures from Saccharomyces cerevisiae, 
Sacch. pastorianus, and Sacch. ellipsoideus on liquid 
wort. After 24 hours' incubation transfer to all media 
and study these veasts in the same manner as described 
as to gas formation, film and sediment formation, spore 
formation, etc. 



LABORATORY GUIDE IN BACTERIOLOGY 
EXERCISE 7. EXAMINATION OF BREWER'S YEAST 

1. Secure some brewer's yeast. 

2. Inoculate a tube of liquid wort and incubate at 
25 C. 

3. Prepare pure cultures from this mixture accord- 
ing to Hansen's method. 

4. When pure cultures have been obtained, study 
these in the same manner as previously directed. 

SECTION 4 
EXAMINATION OF BAKER'S YEAST 

1. Prepare 5 flasks with 100 c.c. sterile water. 

2. Secure a cake of pressed yeast. 

3. Prepare a suspension of i gram in 100 c.c. 
sterile water. 

4. Weigh out about one gram, evaporate on a water 
bath, place in a calcium chlorid desiccator over night, 
weigh again, and determine the percentage of moisture. 

5. Remove 10 c.c. water from a flask containing 
loo c.c. sterile water, and replace with 10 c.c. from the 
suspension. 

6. Transfer i c.c. of the suspension to another dilu- 
tion flask. 

7. Transfer i c.c. from the second flask to a third 
dilution flask. The dilutions prepared are 1:100, 
i : 1,000, i : 10,000, 1:100,000, not considering the 
amount cf moisture in the gram of yeast suspended. 

8. Pour meat extract agar and wort agar plates 
from all dilutions. 

9. Incubate the meat agar plates at 37 C. and the 
wort agar plates at 25 C. 



MOLDS, YEASTS, TORULAE, AND BACTERIA IQI 

10. After 28 hours count the number of colonies 
of bacteria and yeasts present on both plates. Cal- 
culate the numbers in a gram of dry yeasts. 

Bacteria do not multiply well on wort agar, the acid 
reaction being unfavorable. The count on meat agar 
will, therefore, be higher than on wort agar. 

11. Examine the yeast colonies under the micro- 
scope, make stains, examine in water, and transfer the 
different colonies to wort tubes. 

12. Study the cultures prepared as before. 

SECTION 5 
EXAMINATION OF YEAST OF SALT-RISING BREAD 

1. Make smears and stains from the yeast. 

2. Inoculate litmus milk and incubate at 37 C. for 
two or three days. 

3. Make gentian violet and Gram stains, and note 
the dark red color of the litmus. 

4. Inoculate a flask containing 250 c.c. sterile milk 
and incubate for seven days at 37 C. 

5. Determine the amount of acid formed from day 
to day with i/2oNNaOH and phenolphthalei'n as 
indicator. Record the results. 

6. Plate in meat extract agar and wort agar. 

7. Study the colonies in both plates. 

Meat extract agar is not suitable for the organism 
active in this yeast and probably no colonies will 
appear. Beerwort agar is more suitable. The active 
organism is a large bacillus, which forms an amount 
of lactic acid often as high as 3 per cent. When used 
for baking this acid combines with the soda added to 



IQ2 LABORATORY GUIDE IN BACTERIOLOGY 

salt-rising bread dough, and carbon dioxid is liberated, 
which causes the bread to rise. 



SECTION 6 
A STUDY OF TORULAE 

Experiment i 

1. Transfer from laboratory cultures to wort agar. 

2. Make stains, and transfer to all the other media. 

3. Make descriptions of these organisms, and note 
the differences from yeasts. 

Experiment 2 

1. Prepare three plates of wort agar. 

2. Expose these to t^e air in different places. 

3. Incubate at 25 C. 

4. After 48 hours fish ror torula colonies, transfer 
these to all the media, prepare stains, and describe the 
cultures. Inoculate fermentation tubes with carbo- 
hydrates, and inoculate gypsum blocks. Some torulae 
produce gas from lactose; the true yeasts do not, 
Torulae do not form spores. 

SECTION 7 

A STUDY OF ACETIC-ACID BACTERIA 
EXERCISE I 

1. Secure some old vinegar with a film on top. 

2. Prepare a suspension of the film in sterile water. 

3. Plate out in wort agar, and incubate at 25 C. 

4. Transfer the colonies to slant agar. 

5. Inoculate flasks containing about 50 c.c. wort and 
i per cent alcohol with the cultures. 



MOLDS, YEASTS, TORULAE, AND BACTERIA 193 

6. Incubate one flask at room temperature, one at 
37 C, and one at 40 C. 

7. Examine and note the degree of film formation 
at the different temperatures and the differences in 
morphology. 

8. Add small amounts of alcohol from time to time. 

9. Titrate daily with 1/20 N - NaOH and determine 
the amount of acid produced. 

EXERCISE 2 

Inoculate liquid wort to which 3 per cent saccharose 
has been added with an alcohol-forming yeast. After 
alcohol has been produced, inoculate with one of the 
acetic-acid cultures and note the amount of acid 
produced. 



APPENDIX 



APPENDIX 

DILUTION TABLES FOR AGGLUTINATION 



Number 


Amount of Serum 


Amount of Salt 
Solution 


Final Dilution 




i part 


g parts 


1:10 




i part of No. i 


9 parts 


i : 100 




i part of No. 2 


9 parts 


i : i, ooo 




i part of No. 3 


9 parts 


i : 10,000 











Amount of Serum or Serum Dilution 


Suspension 


Final Dilution 


2 parts clear serum 


1 8 parts 


: 10 


i part clear serum 


19 parts 


: 20 




15 parts 


40 


4 parts dil., No. i 


1 6 parts 


So 


2 ? oarts dil No. i . . 


17.5 parts 


:8o 




1 8 parts 




i part dil No i 


19 parts 


: 200 


4 parts dil., No. 2 
2 parts dil., No. 2 
i part dil., No. 2 
4 parts dil., No. 3 


1 6 parts 
i 8 parts 
19 parts 
1 6 parts 
i 8 parts 


1500 
: 1,000 
: 2,000 
: 5,000 
: 10,000 




19 parts 


20 ooo 




i 6 parts 






i 8 parts 


: 100,000 




19 parts 











DILUTION TABLE FOR WATER OR MILK ANALYSIS 



Number 


Amount of Dilution 


Amount of 
Sterile Water 


Final Dilution 




Original 




:i 




IQC.C. of No. i 


9OC.C. 


:io 




ic.c. of No. i 


99C.C. 


: 100 


4 

i;;::::::::::; 


20C.C. Of No. 3 

loc.c. of No. 3 
SG.C. of No. 3 


Soc.c. 
goc.c. 
95C.C. 


:soo 
: 1,000 
: 2,000 




2C.C. Of No. 3 


98c.c. 


: 5,000 


8 . . 


ic.c. of No. 3 


99C.C. 


: 10,000 




5C.c. of No. 4 


95C.C. 


: 20,000 




2C.C. Of No. 4 


98c.c. 


: 50,000 




ic.c. of No. 4 


99C.C. 


: 100,000 




5c.c. of No. 7 


9SC.C. 


: 200,000 




2c.c. of No. 7 


98c.c. 


: 500,000 




ic.c. of No. 7 


99C.C. 


: 1,000,000 











197 



198 LABORATORY GUIDE IN BACTERIOLOGY 



COMPARATIVE TABLES OF WEIGHTS AND MEASURES 

inch =2.54 centimeters 
foot = 0.3048 meters 
yard = 0.9144 meters 
mile = i . 61 kilometers 
micromillimeter (micron, /*) = o.oooooi meters = 0.00003937 in. 
millimeter = o.ooi meters = 0.03937 in. 
centimeter = o.oi meters = 0.3937 in. 
decimeter = o.i meters = 3-937 in- 

meter = i meters= 39.37 in. =3. 28 feet = 1.0936 yds. 

kilometer = 1,000 meters = 3,281 feet =1093. 6 yds. 

i grain = 0.0648 grams 

i ounce, avoirdupois = 28.35 grams 

i ounce, troy = 31.10 grams 

i pound, avoirdupois = 453.6 grams 

i pound, troy = 37 3 



gram = 15. 43 grains 

kilogram = 2 . 205 pounds, avoirdupois 

kilogram = 2.679 pounds, troy 

gallon, U.S. liquid = 3785 cubic centimeters 

quart, U.S. liquid = 946 " cubic centimeters 

pint, U.S. liquid = 473 cubic centimeters 

i ounce, U.S. liquid = 29.57 cubic centimeters 

1,000 cubic centimeters = 1.057 U.S. liquid quarts 



CENTIGRADE AND FAHRENHEIT 


C. 


F. 


C. 


F. 


-18 


-0.4 


- 6 


21.2 


-17 


1-4 


- 5 


23.0 


-16 


3-2 


4 


24.8 


-is 


5-0 


- 3 


26.6 


-14 


6.8 


2 


28.4 


-13 


8.6 


I 


30.2 


12 


10.4 





32-0 


II 


12.2 


I 


33-8 


IO 


14.0 


2 


35-6 


- 9 


IS-8 


3 


37-4 


- 8 


I 7 .6 


4 


39-2 


- 7 


19.4 


5 


41.0 



THERMOMETERS 

C. F. 

6 42.8 

7 44-6 

8 46.4 

9 48.2 

10 50.0 

11 51.8 

12 53-6 

13 55-4 

14 57-2 

15 59-o 

16 60.8 

17 62.6 



APPENDIX IQ9 



c. 


F. 


C. 


F. 


C. 


F. 


18 


64.4 


46 


114.8 


74 


165.2 


19 


66.2 


47 


116.6 


75 


167.0 


20 


68.0 


48 


118.4 


76 


168.8 


21 


69.8 


49 


I2O. 2 


77 


170.6 


22 


71.6 


So 


122. 


78 


172.4 


23 


73-4 


Si 


123.8 


79 


174.2 


24 


75-2 


52 


125.6 


80 


176.0 


25 


77-0 


53 


127.4 


81 


177.8 


26 


78.8 


54 


129.2 


82 


179.6 


27 


80.6 


55 


I3I.O 


83 


181.4 


28 


82.4 


56 


132.8 


84 


183.2 


29 


84.2 


57 


134.6 


85 


185.0 


30 


86.0 


58 


136.4 


86 


186.8 


31 


87.8 


59 


138.2 


87 


188.6 


32 


89.6 


60 


I4O.O 


88 


190.4 


33 


91.4 


61 


I4I.8 


89 


192.2 


34 


93-2 


62 


143.6 


90 


194.0 


35 


95-o 


63 


145-4 


9i 


195.8 


36 


96.8 


64 


147.2 


92 


197.6 


37 


98.6 


65 


149.0 


93 


199.4 


38 


100.4 


66 


150.8 


94 


2OI.2 


39 


IO2.2 


67 


152.6 


95 


203.0 


40 


104.0 


68 


154-4 


96 


204.8 


4i 


IOS.8 


69 


156.2 


97 


206.6 


42 


IO7.6 


70 


158.0 


98 


208.4 


43 


109.4 


7i 


159.8 


99 


2IO.2 


44 


III. 2 


72 


161.6 


IOO 


212. 


45 


II3.0 


73 


163.4 







200 LABORATORY GUIDE IN BACTERIOLOGY 

Endo's Medium 

Pepton (Witte) 10 grams 

Extract of meat 3 grams 

Nad 5 grams 

Distilled water 1,000 c.c. 

Dissolve by boiling. Titrate to o . 2 per cent acid 
to phenolphthalein. Place in autoclave at 15 pounds 
pressure for 6 minutes. Cool and filter off precipitate. 
Titrate again to 0.2 to 0.3 per cent acid. 

Then add: 

Agar threads 30 grams 

Boil until dissolved and make up to 1,000 c.c. 

Saturated solution of basic fuchsin 3 c.c. 

Sodium sulphite solution (5 per cent 

anhydrous) 23 . 3 grams 

Lactose (dissolved in 50 c.c. hot water) 10 grams 

Tube and sterilize in autoclave for 10 minutes at 
10 pounds pressure. 

Russell's Medium 

Make up 1,000 c.c. broth (meat extract) and make 
reaction o . 9 per cent acid to phenolphthalein. Auto- 
clave and filter. Add 15 grams thread agar and 
dissolve by boiling. 

Add i per cent litmus solution in proportion of 
5 per cent. 

Add sufficient n.Na 2 CO 3 to bring the solution to 
the neutral point of the litmus (12-15 c.c.). 

Add i per cent lactose and o . i per cent dextrose. 

Tube and sterilize for 10 minutes at 10 pounds 
pressure. 

The amount of Na 3 CO 3 must be accurate, as other- 
wise the medium will turn greenish after sterilizing. 



INDEX 



INDEX 



Abnormal fermentation in milk, 
159- 

Acetic-acid bacteria, 192. 

Acid broth, 41. 

Acid fermentation in milk, 155. 

Acid-proof bacilli, 131, 132. 

Actinomyces asteroides, 134. 

Actinomyces bovis, 134. 

Actinomyces group, 134. 

Actinomyces hominis, 134. 

Aesculin bile-salt agar, 33. 

Agar-agar, 20. 

Agar: beerwort, 32; bile-salt, 33; 
blood, 40; dextrose, 26; gly- 
cerin, 36; litmus dextrose, 35; 
litmus lactose, 35; mannit, 36; 
slants, inoculation of, 72; syn- 
thetic for plating, 38; standard 
method for preparing, 30; whey, 
36. 

Agglutination test, 118. 

Air, bacterial examination of, 
87; number of bacteria in, 87. 

Albuminoid, 62. 

Alcohol formation by yeasts, 187. 

Alum carmin, 44; hematoxylin, 
44- 

Amido compounds formed by 
soil bacteria, 170. 

Ammonia, formed by soil bacteria, 
171. 

Ammonification, pepton solution 
for, 40. 

Amylase, 63. 

Amylolytic: action of molds, 
184; enzym, 62. 

Anaerobes: In milk, 158; in 
sewage, 149. 

Anaerobic: bacilli, 134; cultiva- 
tion, 134; Buchner's method, 
136; in hydrogen gas, 136; 



Park's method, 135; Wright's 
method, 135. 

Analysis of gas in fermentation 
tubes, 81. 

Anilin gentian violet, 43. 

Anthrax group, 127. 

Arnold steam sterilizer, 12. 

Asiatic cholera, spirillum of, 133. 

Aspergillus oryzae, 184. 

Assimilation of atmospheric nitro- 
gen, 175. 

Autoclave, 14. 

Autopsy, 107. 

Bacilli: acid-proof, 131; anaerobic, 
134. 

Bacillus: ae*rogenes, 112; anthra- 
cis, 127; botulinus, 134; chau- 
vei, 134; suipestifer, 101, 112, 
115; cloacae, 101, 121; coli, 
95, 101, 112; coli anae'ro- 
genes, 112; coli and strepto- 
cocci in milk, 157; coli and 
streptococci in water, 86, 147, 
149; cuniculicida, 127; cyano- 
genes, 159; der Rinderpest, 
127; der Schweineseuche, 127; 
diphtheriae, 124; dysenteriae, 
ii2, 117; edema tis, 134; enter- 
itidis, 112, 115; fecalis alcali- 
genes, 101, 112, 117; of Gart- 
ner, 115; hofmannii, 124; icter- 
oides, 112; influenzae, 133; 
leprae, 131; mallei, 132, 133; 
melitensis, 132; Holler's grass, 
131; capulatus, 123; of bubonic 
plague, 127; of rabbit septi- 
cemia, 127; paratyphosus, 
112, 115; pestis, 127; prodi- 
giosus, 97, 159; pyocyaneus, 
97; rhinoscleromatis, 123; smeg- 
mae, 131; subtilis, 127; tetani, 
134; tuberculosis, 131; typho- 
sus, 95, 112, 117, 150; typho- 
sus in water, 150; violaceus, 



203 



204 LABORATORY GUIDE IN BACTERIOLOGY 



97; viscosus, 159; welchii, 134; 
xerosis, 124, 126. 

Bacteria: and spores in soil, 165; 
chromogenic, 97; from the air, 
87; intestinal, 101; legume, 
176; nitrate, 174; nitrite, 171, 
172; number in air, 87. 

Bacterial examination: of air, 
87; of milk, 88, 151; of soil, 
163; of water, 84, 141. 

Bacteriological technic, i. 

Baker's yeast, 190. 

Beerwort: agar, 32; gelatin, 32; 
hopped, 32; liquid, 32; media, 
32. 

Berkefeld filter, 10, 92. 

Bile-salt agar, 33; broth, 33. 

Bismarck brown, 44. 

Blastomyces derma titidis, 132. 

Blood agar, 40. 

Blood serum, 31. 

Bottcher's moist chamber, 183. 

Bouillon, 1 8. 

Bread paste medium, 41. 

Brewer's yeast, 190. 

Broth: acid, 41; bile-salt, 33; 
calcium carbonate, 41 ; glycerin, 
36; meat, 31; nitrate, 41; 
pepton, 18; sugar-free, 101, 
113; standard method of pre- 
paring, 30. 

Brownian movement, 76. 
Buchner's method of anaerobic 

cultivation, 136. 
Budding of yeasts, 80. 

Calcium carbonate broth, 41. 

Capaldi's egg medium, 37. 

Capaldi's medium, 35. 

Capsulated group, 123. 

Capsule stain, 109; Friedlander's, 
109; Rosenow's, no; Welch's 
no; Welch's modified, 138. 

Carbol fuchsin, 43. 

Carbolic: gentian violet, 45; 
thionin blue, 44. 



Carmin: alum, 44; lithium, 45. 

Casein, 62. 

Chart, culture, 59. 

Cheese, Camembert, 184; molds 
in, 184; Roquefort, 184. 

Cholera red reaction, 130. 

Chromogenic group, 97. 

Cladothrix, 134. 

Cleaning: ot glassware, 8; mix- 
ture, 8. 

Coagulation of milk, 62. 

Coagulative enzym, 62. 

Collecting: micro-organisms from 
the air, 70; molds from the air, 
78, 182; yeasts from the air, 78, 
182; torulae from the air, 78, 
192. 

Collection of water samples, 84, 

145- 

Collodion sacs, 173. 
Colonies, estimation of, 85. 
Colony, 72; counter, 85. 
Comparative table: of Centigrade 

and Fahrenheit thermometers, 

198; of weights and measures, 

198. 

Condensation water, 72, 99. 
Conidia, 79. 
Conradi's medium, 34. 
Cotton alter, 92. 
Counter, colony, 85. 
Cultivation, anaerobic, 134; of 

molds, 182. 
Culture chart, 59. 
Culture media, 18; filtration of, 

21 ; preparation of, 18; reaction 

of, 1 8; titration of, 18. 
Culture of yeasts from one cell, 

187. 
Culture tubes, 9; plugging of, 9; 

potato, 28. 
Cultures, egg, 125; method of 

describing, 53. 

Death-point, thermal, 96. 
Decolorization of litmus milk .63- 



INDEX 



205 



Delafield's hematoxylin, 44. 

Denitrification, solution for test- 
ing, 40. 

Describing cultures, 53. 

Determination: of anaerobes in 
milk, 158; of anaerobes in 
sewage, 149; of B. coli and 
streptococci in milk, 157; of 
B. coli and streptococci in 
water, 86, 147, 149; of bacteria 
and spores in soil, 165; of 
species in water, 86; of spores 
in soil, 165. 

Dextrose: agar, 26; litmus agar, 
35; litmus gelatin, 36; yeast 
water, 33. 

Diastase, 63. 

Differentiation of B. typhosus and 
B. coli, media for, 33. 

Digestion: of blood serum by soil 
bacteria, 168; of casein by soil 
bacteria, 169; of gelatin by 
soil bacteria, 169. 

Dilution tables, 197. 

Diphtheria: antitoxin, 126; ba- 
cillus, 124; group, 124; toxin, 
126. 

Directions for filling out culture 
charts, 58. 

Discontinuous sterilization, n. 

Disinfectants, 94. 

Doane-Buckley method, 161. 

Dog's blood serum, 33. 

Dorset's egg medium, 37. 

Drigalski and Conradi's medium, 
34- 

Dry heat sterilization, n. 

Dunham's pepton solution, 18. 

Dysentery bacillus, 112, 117. 

Effect: of pasteurization of milk, 
88; of sterilization of milk, 88. 

Egg media, 37. 

Ehrlich's anilin gentian violet, 43. 

Endo's medium, 200 

Enriching method of Schottelius, 
130. 



Enzym, 62; amylplytic, 62; 
coagulative, 62; diastatic, 62; 
gelatinolytic, 62; inverting, 
63; production, 62; proteo- 
lytic. 62; rennet, 63. 

Erlenmeyer flask, 7. 

Estimation of colonies, 85. 

Examination: of air, 87; of ice, 
148; of milk, 88, 151; of milk 
for B. coli and streptococci, 157; 
of milk for leukocytes, 160; of 
milk for tubercle bacilli, 155; 
of molds, 184; of rain water, 
148; of sewage, 148; of soil, 
163; of water, 86; of well 
water, 147. 

Exercises on infection and sterili- 
zation, 91. 

Fermentation: abnormal, in milk, 
159; chart, back cover; tube, 
6; tube, gas production in, 
81. 

Filament formation, 129. 

Filling culture tubes, 22. 

Film formation by yeasts, 186. 

Filter: Berkefeld, 10, 92; cotton, 
92; folding of paper, 22. 

Filtration of culture media, 21. 

Finkler and Prior, spirillum of, 
129. 

Flagella stain, 117. 

Formation: of alcohol, 187; of 
amido compounds, 170; of 
ammonia, 171; of film by 
yeasts, 186; of indol, 114, 170; 
of nitrates, 174; of nitrites, 
171; of spores by bacteria, 128; 
of spores by molds, 83; of 
spores by yeasts, 80, 187. 

Formula for determining the 
number of B. coli, streptococci, 
And anaerobes in milk and 
water, 86. 

Fowl cholera, bacillus of, 127. 

Friedlander's capsule stain, 109. 

Friedlander's pneumobacillus, 123. 

Frost's culture chart, 59. 



206 LABORATORY GUIDE IN BACTERIOLOGY 



Fuchsin, carbol, 43. 
Fuller and Johnson's rejuvena- 
tion method, 149. 

Gartner's bacillus, 115. 

Garden earth, inoculation with, 
139- 

Gas: analysis, 81; evolution by 
yeasts, 186; formula, 82; gen- 
erator, 137; production, 81. 

Gelatin, 26; beerwort, 32; dex- 
trose, 36; digestion of, 62; 
liquefaction of, 62; litmus dex- 
trose, 36; litmus lactose, 36; 
pepton, 26; standard methods 
of preparing, 30; whey, 36. 

Gelatinoid, 62. 

Gelatinolytic enzym, 62. 

General bacteriology, 67. 

General directions, 5. 

Gentian violet: anilin, 43; car- 
bolic, 45. 

Germination of mold spores, 83. 

Giltay solution, 40. 

Glanders, 132. 

Glassware: cleaning of, 8; sterili- 
zation of, ii. 

Glycerin: agar, 36; broth, 36; egg 
medium, 37; media, 36. 

Glycerinated potato, 37. 

Glycogen, 113. 

Gonococcus, 109. 

Gonorrheal pus, 109. 

Gram's iodin solution, 43; stain, 
77- 

Granule stain, 124. 

Granules, sulphur, 134. 

Grass bacillus, 131. 

Group: actinomyces, 134; an- 
thrax, i27j capsulated, 123; 
chromogenic, 97; colon, colon- 
typhoid, 112; diphtheria, 124; 
enteritidis, 112-15; hemorrhagic 
septicemia, 127; hog cholera, 
112-15; intermediate, 112-15; 
of acid-proof bacilli, 131; of 
anaerobic bacilli, 134; proteus, 



12 1 ; pyogenic, 105; typhoid- 
dysentery, ii2, 117. 

Groups of bacteria in milk, 162. 

Growing legumes in sand, 178. 

Gruber-Widal test, 118. 

Gypsum blocks, 80. 

Hand lens, 7. 

Hanging drop, 75. 

Hansen's method of pure culture 

of yeasts, 187. 
Hay infusion, 41. 
Heat, influence of, on bacteria, 

94- 
Hematoxylin: alum, 44; Dela- 

field's, 44. 

Hemorrhagic septicemia group, 
127. 

Hesse's medium, 35. 
Hill's test rods, 94. 
Hiss's plating medium, 34; tube 

medium, 34. 

Hog cholera group, 115. 
Hopped beerwort, 32. 
Horse's blood serum, 33. 
Hot-air sterilizer, n. 
Hydrogen gas generator, 137. 

Ice, examination of, 148. 
Impression preparation, 128. 
Incubator, 51. 
Indol, 114; formation of, 114, 

170; test for, 114, 170. 
Infection, phenomena of, 91. 
Influence: of disinfectants, 94; 

of moist heat, 94; of sunlight, 

96. 
Inoculation: of agar slants, 72; 

of animals (see various heads). 
Inspissator, Koch's, 17. 
Intermediate group, 112, 115. 
Intermittent sterilization, n. 
Intestinal group of bacteria, 112. 
Intramuscular inoculation, 131. 
Intraperitoneal inoculation, 123. 



INDEX 



207 



Intravenous inoculation, 106. 

Inverting enzym, 63. 

Involution forms, 117, 121. 

lodin: solution, Gram's, 43; 
starch test, 172. 

Isolation: of B. typhosus in 
water, 150; of legume bacteria, 
176; of nitrate bacteria, 175; 
of nitrite bacteria, 172; of 
unknown bacteria, 139. 

Jar, Novy, 137. 

Jordan's non-protein medium, 42. 

Klatschpraparat, 128. 
Koch's inspissator, 17. 
Kiihne's methylene blue, 45. 

Laboratory rules, 3. 

Lactalbumin, 62. 

Legume, bacteria, 176, 

Leptothrix, 134. 

Leukocytes in milk, 160. 

Liquefaction of blood serum, 57; 
of casein, 62; of gelatin, 62. 

Liquid beerwort, 32. 

Lithium carmin, 45. 

Litmus: decolorization of, 63; 
dextrose agar, 35; dextrose 
gelatin, 36; lactose agar, 35; 
lactose gelatin, 36; mannit 
agar, 36; milk, 27; solution, 35; 
whey, 36. 

Loffler's flagella stain, 117. 

Loffler's methylene blue, 43. 

MacConkey's bile salt agar, 33. 

MacConkey's bile salt broth, 33. 

Malachite green media, 35. 

Maltose, 63. 

Mannit agar, 36. 

Measures and weights, 198. 

Meat: broth, 31; press, 31. 

Media: adjusting reaction of, 18; 
beerwort, 32; culture, 18; egg, 
37; filling in tubes of, 22; 



filtering, 21 ; for differentia- 
tion of B. typhosus and B. 
coli, 33; for the study of soil 
bacteria, 37; for water and 
milk examination, 35; glycerin, 
36; miscellaneous, 40; nitrate, 
41; non-protein, 42; phenol, 
34; preparation of, 18; reaction 
of, 18; standard method of pre- 
paring, 30; synthetic, 42; 
titration of, 18; whey, 36. 

Medium: bread paste, 41; Capal- 
di's, 35; Dorset's egg, 37; 
Drigalski and Conradi's, 34; 
Endo's, 33; glycerin egg, 37; 
Hesse's, 35; Hiss's plating, 34; 
Hiss's tube, 34; Jordan's non- 
protein, 42; ox bile, 35; Uschin- 
sky's, 42; wine must, 41. 

Meningococcus, 133. 

Method: of describing cultures, 
53; of making Gram stain, 77; 
of making plates, 98; of mak- 
ing stained preparations, 76; 
of microscopic examination of 
molds, 79; of preparing agar, 
standard, 30; of preparing 
broth, standard, 30; of pre- 
paring gelatin, standard, 30. 

Methylene blue: Kuhne's, 45; 
Loffler's, 43. 

Micrococcus gonorrheae, 109; 
meningitidis, 133; tetragenous, 
105; zymogenes, 109. 

Micro-organisms from the air, 70. 

Microscope, 45. 

Milk: abnormal fermentation in, 
159; acid fermentation in, 
155; acid-proof bacilli in, 89; 
anaerobes in, 158; bacterial 
examination of, 88; B. coli and 
streptococci in, 157; coagula- 
tion of, 63; examination of, 
for tubercle bacilli, 89; litmus, 
27; leukocytes in, 160; molds 
and yeasts in, 160; pasteur- 
ized, 89, 158; reaction of, 62; 
sterilized, 158; sugar, 62. 

Miscellaneous: media, 40; organ- 
isms, 132. 



208 LABORATORY GUIDE IN BACTERIOLOGY 



Moller's grass bacillus, 131. 

Moller's spore stain, 129. 

Moist chamber, Bottcher's, 183. 

Mold spores, germination of, 83. 

Molds, 182; amylolytic action of, 
184; in cheese, 184; in milk, 
1 60; microscopic examination 
of, 184; yeasts, torulae, and 
acetic-acid bacteria, 179. 

Molecular movement, 75. 

Mouse: holder, in; microscopic 
examination of, xxz. 

Muscle sugar, 101. 

Must, wine, 41. 

Natural souring of milk, 156. 

Needles, platinum, 6. 

Negative Gram stain, 78. 

Neutral- red agar, 33. 

Nicole's carbolic gentian violet, 
45 ; carbolic thionin blue, 44. 

Nitrate: bacteria, 174; broth, 
41; formation, 174; media, 41; 
solution, 41. 

Nitrates, test for, 175. 

Nitrite: bacteria, 171, 172; for- 
mation, 171. 

Nitrites, test for, 114. 

Nitrogen, assimilation of, 175. 

Nitroso-indol reaction, 130. 

Nocardia, 134. 

Non-protein media, 42. 

Normal solution, 20. 

Novy jar, 137. 

Ox-bile medium, 35. 

Ox-blood serum, 33. 

Parietti's solution, 34. 

Park's method of anaerobic culti- 
vation, 135. 

Pasteurizing milk, 89, 158. 

Pedesis, 76. 

Pepton: broth, 18; gelatin, 26; 
solution, Dunham's, 18, solu- 
tion for ammonification, 40. 

Peptonization, 62, 168. 



Petri dishes, 7. 

Phenol: media, 34; sulphonic acid, 
175- 

Phenolphthalein, 19. 

Phenomena: of infection, 91; of 
sterilization, 91. 

Pigments, 100. 

Plate cultures, 98. 

Plating medium, Hiss's, 34. 

Plugging culture tubes, 9. 

Pneumobacillus, 123. 

Pocket inoculation, 125. 

Polar staining, 116, 127. 

Positive Gram stain, 78. 

Potato, 28; glycerinated, 37; 
substitute for, 29; tube, 28. 

Preparation: Gram, 77; hang- 
ing drop, 75; impression, 128; 
of agar-agar, 20; of blood 
agar, 40; of blood serum, 31; 
of bouillon, 18; of broth, 18, 
31; of culture media, 18; of 
dextrose agar, 26; of Dunham's 
pepton solution, 18; of egg 
media, 37; of gelatin, 26; of 
glycerin media, 36; of litmus 
milk, 27; of media for soil ex- 
amination, 37; of media for 
water and milk examination, 35; 
of non-protein media, 42; Pres- 
cott and Breed's method, 161; 
of silica jelly, 38; of soil samples, 
166; of soil suspensions, 166; of 
staining solutions, 43; of sugar- 
free broth, 10 1 ; of whey media, 
36; stained, 76. 

Proteolysis, 62. 

Proteolytic enzym, 62. 

Proteus: group, 121; vulgaris, 
121 ; zenkeri, 121. 

Pure culture: of bacteria, 187; 
of yeasts, 187. 

Pus, gonorrheal, 109. 

Pyocyanin, 100. 

Pyogenic group, 105. 

Rabbit septicemia, 127. 
Rain water, 148. 



INDEX 



209 



Raulin's solution, 42, 185. 
Reaction: indol, 114, 170; nitrites; 

114, 170; nitroso-indol, 130; 

of bacteria on neutral red 

broth, 150; of culture media, 

1 8.; of milk, 62. 
Reduction of nitrates, 177. 
Rejuvenation of cultures, 149. 
Rennet enzym, 62. 
Rinderpest, 127. 
Roquefort cheese, 184. 
Rosenow's capsule stain, no. 
Russell's medium, aoo 

Saccharomyces: cerevisiae, 78, 
1 80; ellipsoideus, 189; pas- 
torianus, 189. 

Sacs, collodion, 173. 

Safranin, 44. 

Samples of water, collection of, 
84. 

Salt rising bread, 191. 

Sarcina lutea, 97, 159. 

Scheme for routine study, 50. 

Schottelius enriching method, 130. 

Schweineseuche, 127. 

Sewage: anaerobes in, 149; con- 
tamination of, 86; examination 
of, 148. 

Sketches of streak and stab 
cultures, 64, 65. 

Silica jelly, 38. 

Snow, examination of, 148. 

Soil: bacteria and spores in, 165; 
examination of, 163; samples, 
165; samplers, 165. 

Solution: Dunham's pepton, 18; 
for assimilation of atmospheric 
nitrogen, 39; for denitrification 
of nitrates, 40; for formation of 
nitrates, 39; for formation of 
nitrites, 38; Giltay, 40; Gram's 
iodin, 43; litmus, 35; nitrate, 
41; normal, 20; Parietti's, 34; 
pepton for ammonification, 40; 
Raulin's, 42; Winogradsky's, 37. 

Solutions, staining, 43. 



Species determination in water, 
86. 

Spirillum: cholerae, 133; group, 
129; metchnikovii, 129; of 
asiatic cholera, 133; of Finkler 
and Prior, 129; tyrogenum, 129. 

Spore: formation of yeasts, 80, 
187; staining, 128. 

Spores: germination of, 83; in 
soil, 1 68; of bacteria, 128; 
of molds, 83; of yeasts, 80. 

Stain: flagella, 117; Friedlander's 
capsule, 100; Gram, 77; Loffler's 
flagella, 109; Pappenheim, 45; 
Rosenow's capsule, no; spore, 
128; Welch's capsule, no; 
Welch's capsule, modified, 138. 

Stained preparation, 76. 

Staining: acid-proof bacilli, 132; 
capsules, 109; solutions, 43. 

Standard method: for preparing 
agar, 30; for preparing broth, 
30; for preparing gelatin, 30. 

Staphylococcus: albus, 105; au- 
reus, 95, 100, 105. 

Starch iodin test, 172. 

Steam sterilizers, u. 

Sterilization, 10; by chemicals, 
n; by dry heat, 11; by moist 
heat, ' n ; discontinuous, n; 
intermittent, n; of blood 
serum, 16; of glassware, n; 
and pasteurization of milk, 88, 
158; of milk, 89, 158; phe- 
nomena of, 91. 

Sterilized milk, 158. 

Sterilizer: Arnold steam, 12; 

hot-air, n. 
Streptococci: in milk, 157; in 

water, 86. 
Streptococcus: lacticus, 100; py- 

ogenes, 105. pneumeae, 109. 
Study: of acetic-acid bacteria, 

192; bacterial, of milk, 88, 162; 

of molds, 83; of molds, yeasts, 

and torulae, 78; of pigments, 

xoo; of soil bacteria, 165; of 

yeasts, 79- 
Subcutaneous injection, 116. 



210 LABORATORY GUIDE IN BACTERIOLOGY 



Substitute for potato, 29. 
Sugar-free broth, 101. 
Sulphur granules, 134. 
Sunlight, influence of, 98. 
Surface water, 146. 
Suspension of soil, 166. 
Synthetic: agar for plating, 38; 
media, 42. 

Table: of Centigrade and Fah- 
renheit thermometers, 198; of 
weights and measures, 198. 

Tables, dilution, 197. 

Technic, bacteriological, i. 

Test: agglutination, 118; for 
indol, 114, 170; for indol and 
nitrites, 114, 170; for nitrates, 
175; for nitrites, 114; Gruber- 
Widal, 118; rods, Hill's, 94; 
starch iodin, 172; Widal, 118. 

Thermal death-point, 96. 

Thermostat, 51. 

Thionin blue, 44. ^ . 

Titration of media, 18. 

Torula amara, 159. 

Torulae, 78, 192. 

Trichomycetes, 134. 

Tube: culture, 9; fermentation, 
7; medium, Hiss's, 34; potato, 
28. 

Tubercle: bacilli in milk. 89; 
bacteria, 175. 



Typhoid-dysentery group, 112, 
117. 

Ultramicroscopic bacteria, 92. 
Uschinsky's medium, 42. 

Water: and milk examination, 
media for, 35; and sewage, 
bacterial examination of, 84, 
141; bacterial examination of, 
84; bath, 70; of condensation, 
72; samples, collection of, 84; 
species determination in, 86. 

Weights and measures, 198. 

Welch's capsule stain, no; modi- 
fied, 138. 

Well water, 147. 

Whey, 62; agar, 36; gelatin, 36; 
litmus, 36; media, 36; 

Widal test, 118. 

Wine must, 41. 

Winogradsky's solution, 37. 

Yeast: baker's, 190; brewer's, 
190; of salt-rising bread, 191; 
water, 32; water, dextrose, 
33- 

Yeasts, 78, 185; alcohol forma- 
tion, 187; budding of, 80; 
film formation of, 186; from 
the air, 78, 185; gas evolution 
of, 1 86; spore formation of, 
80, 187. 

Ziehl-Neelsen's carbol fuchsin, 43. 



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